Patent Grant
9523088
This application contains a Sequence Listing, which has been submitted via EFS, in compliance with 37 C.F.R. §1.52(e), and is hereby incorporated by reference in its entirety. The sequence listing text file submitted via EFS contains the file “30963-US-DIV_SequenceListing.txt” created on Jun. 12, 2014, which is 94,530 bytes in size.
This invention provides modified polynucleotides encoding modified proteases, and methods for altering the production of proteases in microorganisms. In particular, the present invention relates to methods for altering the expression of proteases in microorganisms, such as Bacillus species. The invention discloses modified polynucleotides, vectors, modified polypeptides, and processes for enhancing the production of proteases.
Microorganisms, such as the Gram-positive microorganism that are members of the genus Bacillus, have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into their culture media. Secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media. Secretion of polypeptides into periplasmic space or into their culture media is subject to a variety of parameters, which need to be carefully considered in industrial fermentations.
Indeed, secretion of heterologous polypeptides is a widely used technique in industry. Typically, cells are transformed with a nucleic acid encoding a heterologous polypeptide of interest to be expressed and secreted to produce large quantities of desired polypeptides. This technique has been used to produce large quantities of polypeptides. Expression and secretion of desired polypeptides has been controlled through genetic manipulation of the polynucleotides that encode the desired proteins. Despite various advances in protein production methods, there remains a need in the art to provide efficient methods for extracellular protein secretion.
This invention relates to modified polynucleotides encoding modified proteases, and methods for altering the production of proteases in microorganisms. In particular, the present invention relates to methods for altering the production of proteases in microorganisms, such as Bacillus species. The invention discloses modified polynucleotides, vectors, modified polypeptides, and processes for enhancing the production of proteases.
The present invention relates to polynucleotides, polypeptides and cells that have been genetically manipulated to enhance the production of modified proteins. In particular, the present invention relates to Gram-positive microorganisms having exogenous nucleic acid sequences introduced therein and methods for producing proteins in such host cells, such as members of the genus Bacillus. More specifically, the present invention relates to the production of proteases and to cells that have been genetically manipulated to have an altered capacity to produce the expressed proteins. In particular, the present invention provides for the enhanced production of proteases by a microorganism.
In some embodiments, the present invention provides for isolated modified polynucleotides encoding modified full-length proteases wherein the portion of the polynucleotide sequence that encodes the pro region of the full-length precursor protease comprises mutations encoding substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and 244. In some embodiments the modified full-length protease is a serine protease, wherein the portion of the polynucleotide sequence that encodes the pro region of the full-length precursor protease comprises mutations encoding substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and 244. In other embodiments, the full-length protease is an alkaline serine protease derived from a wild-type or variant precursor alkaline serine protease, wherein the portion of the polynucleotide sequence that encodes the pro region of the full-length precursor protease comprises mutations encoding substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and 244. In yet other embodiments, the full-length protease is an alkaline serine protease derived from a wild-type or variant precursor alkaline serine protease that is a B. Clausii or a B. lentus alkaline serine protease, wherein the portion of the polynucleotide sequence that encodes the pro region of the full-length precursor protease comprises mutations encoding substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and 244. In some embodiments, the isolated modified polynucleotide comprises the polynucleotide sequence set forth in SEQ ID NOS:1, 9 or 240.
In some embodiments, the present invention provides for isolated modified polynucleotides encoding modified full-length proteases wherein the portion of the polynucleotide sequence that encodes the pro region of the full-length precursor protease comprises mutations encoding substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and 244, wherein the at least one substitution is made at one amino acid position chosen from E33, E43, A44, E47, V49, E57, A59, E63, E70, E74, E84, and E88 of SEQ ID NO:5, 13 or 244. In some other embodiments, the amino acid substitution of E33 is chosen from E33D, E33I, E33S, E33N, E33K, E33H, E33Q and E33R. In yet other embodiments, the amino acid substitution of E57 is selected from E57F, E57W, E57K, E57R, E57D, E57M, E57C, E57Q, E57S, E57H and E57N.
In some embodiments, the invention provides isolated modified polynucleotides comprising a precursor pro sequence that has been mutated to encode substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 or 244. In some additional embodiments, the isolated modified polynucleotides further comprise a polynucleotide encoding a full-length protease comprising a mature region, wherein the polynucleotide encodes a mature region of the protease that is at least about 70% identical to SEQ ID NOS:8, 16 or 247.
In some embodiments, the invention provides at least one vector comprising modified polynucleotides encoding modified full-length proteases wherein the portion of the polynucleotide sequence that encodes the pro region of the full-length precursor protease comprises mutations encoding substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and 244, or isolated modified polynucleotides comprising a precursor pro sequence that has been mutated to encode substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 or 244.
In other embodiments, the invention provides a host cell that is transformed with the vector comprising the modified polynucleotides of the invention. In some preferred embodiments, the transformed host cell is a microorganism. For example, in some embodiments, the invention provides a host cell that is transformed with at least one vector comprising modified polynucleotides encoding modified full-length proteases wherein the portion of the polynucleotide sequence that encodes the pro region of the full-length precursor protease comprises mutations encoding substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and 244, or that is transformed with an isolated modified polynucleotides comprising a precursor pro sequence that has been mutated to encode substitutions at least at one amino acid position chosen from positions equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 or 244. In some embodiments, the host cell that is transformed with the polynucleotides of the invention is a microorganism is chosen from the group consisting of Bacillus sp., Streptomyces sp., Escherichia sp. and Aspergillus sp. In some other embodiments, the host cell is B. subtilis.
In some embodiments, the invention provides proteases produced by the transformed host cells of the invention.
The invention also provides methods for producing a heterologous protease in a microorganism, wherein the method comprises the steps of: (a) culturing a Bacillus host cell under suitable conditions, wherein the Bacillus host cell comprises a modified polynucleotide encoding a modified protease; and (b) allowing production of the protease by the microorganism. In some embodiments, the protease produced by the Bacillus host is recovered. Any one of the modified polynucleotides provided herein finds use in the methods of the invention.
In some embodiments, invention provides methods for producing a heterologous alkaline serine protease in a microorganism, wherein the method comprises the steps of: (a) culturing a Bacillus host cell under suitable conditions, wherein the Bacillus host cell comprises a modified polynucleotide encoding a modified protease; and (b) allowing production of the protease by the microorganism. The invention also provides methods for producing a heterologous protease in a microorganism, wherein the method comprises the steps of: (a) culturing a Bacillus host cell under suitable conditions, wherein the Bacillus host cell comprises a modified polynucleotide encoding a modified protease comprising a mature region that is at least about 70% identical to SEQ ID NOS:8, 16 or 247; and (b) allowing production of the protease by the microorganism. In some embodiments, the protease produced by the Bacillus host is recovered.
In some embodiments, the invention provides methods for producing a heterologous protease in a microorganism, wherein the method comprises the steps of: (a) culturing a Bacillus host cell under suitable conditions, wherein the Bacillus host cell comprises a modified polynucleotide encoding a modified protease that comprises a pro region comprising a mutation encoding a substitution at least at one amino acid position chosen from positions that are equivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 or 244.; and (b) allowing production of the protease by the microorganism. In other embodiments, the at least one amino acid substitution is chosen from E33D, E33I, E33S, E33N, E33K, E33H, E33Q, E33R, E57F, E57W, E57K, E57R, E57D, E57M, E57C, E57Q, E57S, E57H and E57N.
The invention also provides methods for producing in a microorganism a heterologous protease that is a B. clausii or a B. lentus protease, wherein the method comprises the steps of: (a) culturing a Bacillus host cell under suitable conditions, wherein the Bacillus host cell comprises a modified polynucleotide encoding a modified protease; and (b) allowing production of the protease by the microorganism.
The invention also provides methods for producing a heterologous protease in a microorganism, wherein the method comprises the steps of: (a) culturing under suitable conditions a Bacillus host cell chosen from B. licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens, B. brevis, B. stearothermophilus, B. clausii, B. alkalophilus, B. halodurans, B. coagulans, B. circulans, B. pumilus, and B. thuringiensis, wherein the Bacillus host cell comprises a modified polynucleotide encoding a modified protease; and (b) allowing production of the protease by the microorganism. In some embodiments, the host cell is a B. subtilis host cell.
The invention also provides methods for producing a heterologous protease in a microorganism, wherein the method comprises the steps of: (a) culturing a Bacillus host cell under suitable conditions, wherein the Bacillus host cell comprises a modified polynucleotide encoding a modified protease; and (b) allowing production of the protease by the microorganism, and wherein the heterologous protease exhibits a ratio of production of at least 1.
This invention relates to modified polynucleotides encoding modified proteases, and methods for altering the production of proteases in microorganisms. In particular, the present invention relates to methods for altering the production of proteases in microorganisms, such as Bacillus species. The invention discloses modified polynucleotides, vectors, modified polypeptides, and processes for enhancing the production of proteases.
The present invention provides modified polynucleotides encoding proteases having a mutated pro region, as well as the modified proteases encoded by the modified polynucleotides, and methods for producing the same. The modified protease polynucleotides are suitable for expressing the modified proteases in microorganisms, and processing the mature forms at levels greater than the corresponding precursor proteases. The produced proteases find use in the industrial production of enzymes, suitable for use in various industries, including but not limited to the cleaning, animal feed and textile processing industry. The present invention also provides means to produce these enzymes. In some preferred embodiments, the proteases of the present invention are in pure or relatively pure form.
Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and recombinant DNA fields, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Second Edition (Cold Spring Harbor), [1989]); and Ausubel et al., “Current Protocols in Molecular Biology” [1987]). All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Markham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) provide those of skill in the art with a general dictionaries of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular “a”, “an” and “the” includes the plural reference unless the context clearly indicates otherwise. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.
A “modified protease” is a full-length protease that has an amino acid sequence that is derived from the amino acid sequence of a full-length “precursor protease”. The precursor protease may also be referred to as “unmodified protease”. The modified protease differs from its precursor protease in the pro region. The precursor protease can be a naturally-occurring i.e. wild-type protease, or it can be a variant protease. It is the pro region of the wild-type or variant protease that is modified to generate a modified protease. The amino acid sequence of the modified protease is said to be “derived” from the precursor protease amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the pro region of the precursor amino acid sequence. In preferred embodiments, one or more amino acids of the pro region of the precursor protease are substituted to generate the modified protease. Such modification is of the “precursor DNA sequence” which encodes the amino acid sequence of the precursor protease rather than manipulation of the precursor protease per se. The modified proteases herein encompass the substitution of any of the nineteen naturally occurring amino acids at any one of the amino acid residues of the pro region of the precursor protease. In this context, both “modified” and “precursor” proteases are full-length proteases comprising a signal peptide, a pro region and a mature region. The polynucleotides that encode the modified sequence are referred to as “modified polynucleotides”, and the polynucleotides that encode the precursor protease are referred to as “precursor polynucleotides”. “Precursor polypeptides” and “precursor polynucleotides” can be interchangeably referred to as “unmodified precursor polypeptides” or “unmodified precursor polynucleotides”, respectively.
“Naturally-occurring” or “wild-type” refers to a protease or a polynucleotide encoding a protease having the unmodified amino acid sequence identical to that found in nature. Naturally occurring enzymes include native enzymes, those enzymes naturally expressed or found in the particular microorganism. A sequence that is wild-type or naturally-occurring refers to a sequence from which a variant is derived. The wild-type sequence may encode either a homologous or heterologous protein.
As used herein, “variant” refers to a precursor protein which differs from its corresponding wild-type protein by the addition of one or more amino acids to either or both the C- and N-terminal end, substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, deletion of one or more amino acids at either or both ends of the protein or at one or more sites in the amino acid sequence, and/or insertion of one or more amino acids at one or more sites in the amino acid sequence. A variant protein in the context of the present invention is exemplified by the B. clauisii protease V049 (SEQ ID NO:13), which is a variant of the naturally-occurring protein Maxacal (SEQ ID NO:5). The precursor protein of the variant can be a wild-type or variant protein.
As used herein, “equivalent to,” refers to a residue at the enumerated position in a protein or peptide, or a residue that is analogous, homologous, or corresponding to an enumerated residue in a protein or peptide.
The term “production” with reference to a protease, encompasses the two processing steps of a full-length protease including: 1. the removal of the signal peptide, which is known to occur during protein secretion; and 2. the removal of the pro region, which creates the active mature form of the enzyme and which is known to occur during the maturation process (Wang et al., Biochemistry 37:3165-3171 (1998); Power et al., Proc Natl Acad Sci USA 83:3096-3100 (1986)).
The term “processed” with reference to a mature protease refers to the maturation process that a full-length protein e.g. a protease, undergoes to become an active mature enzyme.
The terms “activity ratio” and “ratio of production” are used interchangeably to refer to the ratio of the enzymatic activity of a mature protease that was processed from modified protease to the enzymatic activity of a mature protease that was processed from an unmodified protease.
The term “full-length protein” herein refers to a primary gene product of a gene and comprising a signal peptide, a pro sequence and a mature sequence.
The term “signal sequence” or “signal peptide” refers to any sequence of nucleotides and/or amino acids which may participate in the secretion of the mature or precursor forms of the protein. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protein gene, which participate in the effectuation of the secretion of protein.
The term “pro sequence” or “pro region” is an amino acid sequence between the signal sequence and mature protease that is necessary for the secretion/production of the protease. Cleavage of the pro sequence will result in a mature active protease. To exemplify, a pro region of a protease of the present invention at least includes the amino acid sequence identical to residues 28-111 of SEQ ID NO:5, 13 or 244.
The terms “mature form” or “mature region” refer to the final functional portion of the protein. To exemplify, a mature form of the protease of the present invention at least includes the amino acid sequence identical to residues 112-380 of SEQ ID NO:5, 13 or 244. In this context, the “mature form” is “processed from” a full-length protease, wherein the processing of the full-length protease encompasses the removal of the signal peptide and the removal of the pro region.
As used herein, the term “heterologous protein” refers to a protein or polypeptide that does not naturally occur in the host cell. Similarly, a “heterologous polynucleotide” refers to a polynucleotide that does not naturally occur in the host cell.
As used herein, “homologous protein” refers to a protein or polypeptide native or naturally occurring in a cell. Similarly, a “homologous polynucleotide” refers to a polynucleotide that is native or naturally occurring in a cell.
As used herein, the term “promoter” refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. In preferred embodiments, the promoter is appropriate to the host cell in which the target gene is being expressed. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also termed “control sequences”) is necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous.
As used herein, the terms “protease,” and “proteolytic activity” refer to a protein or peptide exhibiting the ability to hydrolyze peptides or substrates having peptide linkages. Many well known procedures exist for measuring proteolytic activity (Kalisz, “Microbial Proteinases,” In: Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology, [1988]). For example, proteolytic activity may be ascertained by comparative assays which analyze the produced protease's ability to hydrolyze a commercial substrate. Exemplary substrates useful in such analysis of protease or proteolytic activity, include, but are not limited to di-methyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICN Biomedical 902111). Colorimetric assays utilizing these substrates are well known in the art (See e.g., WO 99/34011; and U.S. Pat. No. 6,376,450, both of which are incorporated herein by reference. The AAPF assay (See e.g., Del Mar et al., Anal. Biochem., 99:316-320 [1979]) also finds use in determining the production of mature protease. This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration.
As used herein, “the genus Bacillus” includes all species within the genus “Bacillus,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus.” The production of resistant endospores in the presence of oxygen is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.
The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length. These terms include, but are not limited to, a single-, double-stranded DNA, genomic DNA, cDNA, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases. Non-limiting examples of polynucleotides include genes, gene fragments, chromosomal fragments, ESTs, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
As used herein, the terms “DNA construct” and “transforming DNA” are used interchangeably to refer to DNA used to introduce sequences into a host cell or organism. The DNA may be generated in vitro by PCR or any other suitable technique(s) known to those in the art. In particularly preferred embodiments, the DNA construct comprises a sequence of interest (e.g., a modified sequence). In some embodiments, the sequence is operably linked to additional elements such as control elements (e.g., promoters, etc.). In some embodiments, the DNA construct comprises sequences homologous to the host cell chromosome. In other embodiments, the DNA construct comprises non-homologous sequences. Once the DNA construct is assembled in vitro it may be used to mutagenize a region of the host cell chromosome (i.e., replace an endogenous sequence with a heterologous sequence).
As used herein, the term “vector” refers to a polynucleotide construct designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, and plasmids. In some embodiments, the polynucleotide construct comprises a DNA sequence encoding the full-length protease (e.g., modified protease or unmodified precursor protease).
As used herein, the term “plasmid” refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in some eukaryotes or prokaryotes, or integrates into the host chromosome.
As used herein in the context of introducing a nucleic acid sequence into a cell, the term “introduced” refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, conjugation, and transduction (See e.g., Ferrari et al., “Genetics,” in Hardwood et al, (eds.), Bacillus, Plenum Publishing Corp., pages 57-72, [1989]).
As used herein, the terms “transformed” and “stably transformed” refers to a cell that has a non-native (heterologous)polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained for at least two generations.
The present invention provides isolated modified polynucleotides encoding amino acid sequences, encoding modified proteases. The modified proteases are obtained by mutating the polynucleotide sequence of precursor proteases. Specifically, one or more mutations of the polynucleotide sequence encoding the pro region of the precursor protease are made to provide the modified polynucleotides of the invention.
This invention relates to modified polynucleotides encoding modified proteases, and methods for altering the production of proteases in microorganisms. In particular, the present invention relates to methods for altering the production of proteases in microorganisms, such as Bacillus species. The invention discloses modified polynucleotides, vectors, modified polypeptides, and processes for enhancing the production of proteases.
In some embodiments, the invention provides modified polynucleotides encoding modified proteases that are derived from wild-type or variant precursor proteases of animal, vegetable or microbial origin. Polynucleotides encoding precursor proteases that are derived from microorganisms are preferred, and comprise polynucleotides encoding wild-type precursor proteases and variant precursor proteases, which have been derived from the wild-type forms. In some embodiments, the modified polynucleotides of the invention are derived from polynucleotides encoding precursor proteases of microbial origin. The invention also encompasses polynucleotides encoding modified proteases that are derived from polynucleotides encoding protein engineered precursors. Polynucleotides encoding serine proteases are the preferred precursor protease polynucleotides, of which the alkaline microbial protease polynucleotides are particularly preferred. Serine proteases are enzymes which catalyze the hydrolysis of peptide bonds in which there is an essential serine residue at the active site. Serine proteases have molecular weights in the approximately 25,000 to 30,000 range (See, Priest, Bacteriol. Rev., 41:711-753 [1977]). In some preferred embodiments, polynucleotides encoding subtilisin and subtilisin variants are preferred precursor serine protease polynucleotides. In some embodiments, the invention encompasses polynucleotides encoding modified proteases that have been derived from polynucleotides of microorganisms such as B. licheniformis, B. subtilis, B. amyloliquefaciens, B. clausii, B. lentus and B. halodurans. A wide variety of Bacillus polynucleotides encoding subtilisins have been identified and sequenced, for example, subtilisin 168, subtilisin BPN′, subtilisin Carlsberg, subtilisin DY, subtilisin 147 and subtilisin 309 (See e.g., EP 414279 B; WO 89/06279; and Stahl et al., J. Bacteriol., 159:811-818 [1984]; each of which is incorporated by reference in their entirety). In some embodiments of the present invention, mutant (e.g., variant) protease polynucleotides serve as precursor polynucleotides encoding proteins from which the modified proteases of the invention are derived. Numerous references provide examples of variant proteases (See e.g., WO 99/20770; WO 99/20726; WO 99/20769; WO 89/06279; RE 34,606; U.S. Pat. No. 4,914,031; U.S. Pat. No. 4,980,288; U.S. Pat. No. 5,208,158; U.S. Pat. No. 5,310,675; U.S. Pat. No. 5,336,611; U.S. Pat. No. 5,399,283; U.S. Pat. No. 5,441,882; U.S. Pat. No. 5,482,849; U.S. Pat. No. 5,631,217; U.S. Pat. No. 5,665,587; U.S. Pat. No. 5,700,676; U.S. Pat. No. 5,741,694; U.S. Pat. No. 5,858,757; U.S. Pat. No. 5,880,080; U.S. Pat. No. 6,197,567; and U.S. Pat. No. 6,218,165; each of which is incorporated by reference in its entirety). In other embodiments, polynucleotides encompassed by the invention include modified polynucleotides that are derived from polynucleotides encoding commercially available proteases. For example, commercially available proteases include, but are not limited to ALCALASE™, SAVINASE™, PRIMASE™, DURALASE™, ESPERASE™, and KANNASE™ (Novo Nordisk A/S), MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, and PURAFECT OXP™, (Genencor International Inc.). In some embodiments, the invention encompasses modified polynucleotides that are derived from polynucleotides encoding precursor proteases from B. clausii. In other embodiments, the invention encompasses modified polynucleotides that are derived from polynucleotides encoding precursor proteases from B. lentus.
In some embodiments, the modified full-length polynculceotides of the invention comprise sequences that encode the pro region of precursor proteases that have been mutated. The polynucleotide sequence encoding the pro region of any suitable precursor protease finds use in the generation of one or more modified polynucleotides of the invention. In some preferred embodiments, the portion of a precursor polynucleotide sequence encoding a pro region is mutated to encode one or more amino acid substitutions at positions that are equivalent to positions 1-109 of the protease of SEQ ID NO:5, 13 or 244. In some embodiments, the modified precursor polynucleotides encode for at least one amino acid substitutions in the pro region at positions equivalent to E33, E43, A44, E47, V49, E57, A59, E63, E70, E74, E84, and E88 of SEQ ID NO:5, 13 or 244. In some embodiments, the polynucleotide sequence encoding the amino acid at position equivalent to E33 is mutated to encode at least one of substitutions E33D, E33I, E33S, E33N, E33K, E33H, E33Q, or E33R. In some other embodiments, the polynucleotide sequence encoding the amino acid at position equivalent to E57 is mutated to encode one of substitutions E57F, E57W, E57K, E57R, E57D, E57M, E57C, E57Q, E57S, E57H, and/or E57N.
In some embodiments, the precursor B. clausii protease polynucleotide (e.g., SEQ ID NO:1) encodes the wild-type protease (MAXACAL™; SEQ ID NO:5).
In other embodiments, the precursor B. clausii protease polynucleotide (e.g. SEQ ID NO:9) encodes a variant protease (e.g., SEQ ID NO:13)
In other embodiments, the precursor protease polynucleotide is a B. lentus polynucleotide (e.g. SEQ ID NO:240), which encodes a protease GG36 of SEQ ID NO:244.
In some embodiments, the present invention provides modified full-length polynucleotides derived from the full-length precursor polynucleotide of SEQ ID NO:9.
The polynucleotide sequence of SEQ ID NO:9 comprises a sequence (SEQ ID NO:10) that, when expressed, is contemplated to encode a signal sequence peptide (SEQ ID NO:14), which spans amino acids 1-27 of SEQ ID NO:13; an N-terminal pro sequence (SEQ ID NO:11) encoding a pro region sequence (SEQ ID NO:15), which spans amino acid residues 28-111 of SEQ ID NO:13; and a mature serine protease sequence (SEQ ID NO:12) encoding amino acid residues 112-380 of SEQ ID NO:13 (i.e., SEQ ID NO:16). The polynucleotide sequence encoding the first 8 amino acids of the signal peptide of the protease of SEQ ID NO: 13 is the sequence encoding the first 8 amino acids of the B. subtilis AprE protease.
In other embodiments, the present invention provides modified full-length polynucleotides derived from the wild-type full-length precursor polynucleotide of SEQ ID NO:1. The polynucleotide of SEQ ID NO:1 comprises a sequence SEQ ID NO:2 that, when expressed, is contemplated to encode a signal sequence peptide (SEQ ID NO:6) which is amino acids 1-27 of SEQ ID NO:5; an N-terminal pro sequence (SEQ ID NO:3), encoding amino acid residues 28-111 of SEQ ID NO:5 (i.e. SEQ ID NO:7); and a wild-type mature serine protease sequence (SEQ ID NO:4) encoding amino acid residues 112-380 of SEQ ID NO:5 (i.e., SEQ ID NO:8)).
In other embodiments, the present invention provides modified full-length polynucleotides derived from the variant full-length precursor polynucleotide of SEQ ID NO:240. The polynucleotide of SEQ ID NO:240 comprises a sequence SEQ ID NO:241 that, when expressed, is contemplated to encode a signal sequence peptide (SEQ ID NO:245) which is amino acids 1-27 of SEQ ID NO:244; an N-terminal pro sequence (SEQ ID NO:242), encoding amino acid residues 28-111 of SEQ ID NO:244 (i.e. SEQ ID NO:246); and a wild-type mature serine protease sequence (SEQ ID NO:243) encoding amino acid residues 112-380 of SEQ ID NO:244 (i.e., SEQ ID NO:247)). The polynucleotide sequence encoding the first 8 amino acids of the signal peptide of the precursor protease of SEQ ID NO: 244 is the sequence encoding the first 8 amino acids of the B. subtilis AprE protease. The pro portion and the mature portion of the precursor GG36 are encoded by the GG36 wild-type sequence.
In preferred embodiments, the modified full-length polynculceotides of the invention comprise sequences that encode the pro region of precursor proteases that have been mutated. The polynucleotide sequence encoding the pro region of any suitable precursor protease finds use in the generation of one or more modified polynucleotides of the invention. In some preferred embodiments, the portion of a precursor polynucleotide sequence encoding a pro region is mutated to encode one or more amino acid substitutions at positions that are equivalent to positions 1-109 of the protease of SEQ ID NO:13. In other embodiments, the substitutions are made at positions that are equivalent to 1-109 of the protease of SEQ ID NO:5. In yet other embodiments, the substitutions are made at positions that are equivalent to positions 1-109 of the protease of SEQ ID NO:244. In some embodiments, the modified precursor polynucleotides encode for at least one amino acid substitutions at positions equivalent to E33, E43, A44, E47, V49, E57, A59, E63, E70, E74, E84, and E88 of SEQ ID NO:5, 13 or 244. In some embodiments, the polynucleotide sequence encoding the amino acid at position equivalent to E33 is mutated to encode at least one of substitutions E33D, E33I, E33S, E33N, E33K, E33H, E33Q, or E33R. In some other embodiments, the polynucleotide sequence encoding the amino acid at position equivalent to E57 is mutated to encode one of substitutions E57F, E57W, E57K, E57R, E57D, E57M, E57C, E57Q, E57S, E57H, and/or E57N.
As discussed above, in some embodiments, the polynucleotide encoding any precursor protease is mutated to generate at least one modified polynucleotide encoding at least one modified protease having a mutated pro region. In some particularly preferred embodiments, full-length polynucleotides encoding serine proteases find use in the generation of the modified polynucleotides of the invention. In some alternative particularly preferred embodiments, full-length polynucleotides encoding alkaline serine proteases find use. As discussed above, the polynucleotides encoding the precursor proteases are from microorganisms including but not limited to B. licheniformis, B. subtilis, B. amyloliquefaciens, B. clausii, B. lentus and B. halodurans. The invention provides for any one of the In some embodiments, the modified polynucleotides of the invention that encode full-length modified proteases comprise sequences that encode mature forms of the proteases that share at least about 65% amino acid sequence identity, preferably at least about 70% amino acid sequence identity, more preferably at least about 75% amino acid sequence identity, still more preferably at least about 80% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, even more preferably at least about 90% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, yet more preferably at least about 95% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, still more preferably at least about 98% amino acid sequence identity, and most preferably at least about 99% amino acid sequence identity with the amino acid sequence of the mature form of the precursor protease and have comparable or enhanced production activity as compared to the precursor polypeptide. In some embodiments, the modified polynucleotides of the invention that encode the full-length modified proteases comprise sequences that encode mature forms of the proteases that share at least about 65% amino acid sequence identity, preferably at least about 70% amino acid sequence identity, more preferably at least about 75% amino acid sequence identity, still more preferably at least about 80% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, even more preferably at least about 90% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, yet more preferably at least about 95% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, still more preferably at least about 98% amino acid sequence identity, and most preferably at least about 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:8, SEQ ID NO:16 or SEQ ID NO:247.
In some embodiments, the modified polynucleotides encode full-length amino acid sequences that share up to 65% amino acid sequence identity, preferably up to about 70% amino acid sequence identity, more preferably up to about 75% amino acid sequence identity, still more preferably up to about 80% amino acid sequence identity, more preferably up to about 85% amino acid sequence identity, even more preferably up to about 90% amino acid sequence identity, more preferably up to about 92% amino acid sequence identity, yet more preferably up to about 95% amino acid sequence identity, more preferably up to about 97% amino acid sequence identity, still more preferably up to about 98% amino acid sequence identity, and most preferably up to about 99% amino acid sequence identity with the amino acid sequence of the precursor protease and have comparable or enhanced production activity, as compared to the precursor polypeptide.
As will be understood by the skilled artisan, due to the degeneracy of the genetic code, a variety of modified polynucleotides encode modified proteases. In some other embodiments of the present invention, polynucleotides comprising a nucleotide sequence having at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 92% sequence identity, at least about 95% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity and at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NOS:4, 12, or 243 are provided.
In some embodiments, the percent identity shared by polynucleotide sequences is determined by direct comparison of the sequence information between the molecules by aligning the sequences and determining the identity by methods known in the art. In some embodiments, the percent identity (e.g., amino acid sequence, nucleic acid sequence, and/or gene sequence) is determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs find use in these analyses, including those described above. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above.
An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol., 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See, Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparison of both strands.
The BLAST algorithm then performs a statistical analysis of the similarity between two sequences (See e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 [1993]). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a serine protease nucleic acid of this invention if the smallest sum probability in a comparison of the test nucleic acid to a serine protease nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Where the test nucleic acid encodes a serine protease polypeptide, it is considered similar to a specified serine protease nucleic acid if the comparison results in a smallest sum probability of less than about 0.5, and more preferably less than about 0.2.
In some embodiments of the present invention, sequences were analyzed by BLAST and protein translation sequence tools. In some experiments, the preferred version was BLAST (Basic BLAST version 2.0). The program chosen was “BlastX”, and the database chosen was “nr.” Standard/default parameter values were employed.
Several methods are known in the art that are suitable for generating modified polynucleotide sequences of the present invention, including but not limited to site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.
In some embodiments, the modified polynucleotide sequences of the invention comprise modifications of the sequence encoding the pro region of the protease that are generated with site directed mutagenesis in at least one codon. In other preferred embodiments, site directed mutagenesis is performed for two or more codons. In some further embodiments, modified polynucleotide sequences that encode the pro region of the modified protease have up to about 40%, up to about 45%, up to about 50%, up to about 55%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 98%, or up to about 99% homology with the polynucleotide that encodes the pro region of the precursor polypeptide e.g. SEQ ID NOS:3, 11 or 242. In some alternative embodiments, the modified polynucleotide is generated in vivo, using any known mutagenic procedure such as, for example, radiation, nitrosoguanidine and the like. The desired modified polynucleotide sequence is then isolated and used in the methods provided herein.
In some preferred embodiments, site saturation mutagenesis of the pro region of the precursor protease polynucleotides is accomplished by using a composition comprising mutagenic primers. Mutagenic primers do not precisely match the precursor polynucleotide, and the mismatch or mismatches in the primers are used to introduce the desired mutation into the template polynucleotide. Non-mutagenic primers, which match precisely the precursor polynucleotide are also included in the primer composition. By adding a mixture of mutagenic primers and non-mutagenic primers corresponding to at least one of the mutagenic primers, a nucleic acid library in which a variety of mutational patterns are presented is produced. For example, if it is desired that some of the members of the mutant nucleic acid library retain their precursor sequence at certain positions while other members are mutant at such sites, the non-mutagenic primers provide the ability to obtain a specific level of non-mutant members within the nucleic acid library for a given residue. The methods of the invention employ mutagenic and non-mutagenic oligonucleotides which are generally between about 10 to about 50 bases in length, more preferably about 15 to about 45 bases in length. With respect to corresponding mutagenic and non-mutagenic primers, it is not necessary that the corresponding oligonucleotides be of identical length, but only that there is overlap in the region corresponding to the mutation to be added.
In some embodiments, primers are added in a pre-defined ratio. For example, if it is desired that the resulting library have a significant level of a certain specific mutation and a lesser amount of a different mutation at the same or different site, by adjusting the amount of primer added, the desired biased library is produced. Alternatively, by adding lesser or greater amounts of non-mutagenic primers, the frequency with which the corresponding mutation(s) are produced in the mutant nucleic acid library is adjusted. Kits comprising compositions of primers for site—directed mutagenesis are commercially available and include the QuikChange® site-directed mutagenesis kit (Stratagene, San Diego, Calif.). In some embodiments, precursor polynucleotides are further modified to encode modified proteases that comprise two or more amino acid substitutions in the pro region. Kits for performing multi-site directed mutagenesis are also commercially available (e.g., QuikChange® Multisite, Stratagene, San Diego, Calif.).
In some embodiments, the modified polynucleotides of the invention are generated using site-saturation methods. In some other embodiments, each of the modified polynucleotides comprises at least one amino acid substitution in the pro region.
The present invention provides modified full-length precursor proteases that are encoded by any one of the modified polynucleotides of the invention. The invention provides for mature forms of proteases that when processed from modified precursor proteases, can be produced at levels that are greater than those levels attained by processing of the unmodified precursor proteases.
The invention encompasses full-length proteases that have been modified by mutating at least one amino acid of the pro region of a precursor protease. In some embodiments, the precursor protease is mutated at one or more amino acids of the pro region that are equivalent to amino acids 28-109 of the pro region (SEQ ID NO:15) of the full-length precursor protease of SEQ ID NO:13, the pro region (SEQ ID NO:7) of the full-length precursor protease of SEQ ID NO:5, or the pro region (SEQ ID NO:246) of the full-length precursor protease of SEQ ID NO:244. In some embodiments, the amino acid substitutions are made at positions equivalent to E33, E43, A44, E47, V49, E57, A59, E63, E70, E74, E84 and/or E88 of the B. clausii V049 precursor protease (SEQ ID NO:13), of the B. clausii wild-type protease (SEQ ID NO:5), or of the wild-type B. lentus protease (SEQ ID NO: 244).
In some embodiments, the substitution of amino acids at positions equivalent to E33, include, but are not limited to E33D, E33I, E33S, E33N, E33K, E33H, E33Q or E33R. In other embodiments, the substitution of E57, includes, but is not limited to E57F, E57W, E57K, E57R, E57D, E57M, E57C, E57Q, E57G, E57S, E57H or E57N. The present invention encompasses modified proteases wherein any one amino acid substitution at each of the amino acid positions equivalent to amino acid 28-109 of SEQ ID NO:5, 13 or 244 is made with any one of the nineteen naturally occurring L-amino acids to enhance the production/secretion of the modified protease in comparison to that of the precursor protease (See, Table 2 and Example 4). The amino acids are referred to herein by the commonly used and understood one letter code (See e.g., Dale, M. W. (1989), Molecular Genetics of Bacteria, John Wiley & Sons, Ltd.).
In some embodiments, the modified precursor protease polypeptides comprise mature regions that share at least about 65% amino acid sequence identity, preferably at least about 70% amino acid sequence identity, more preferably at least about 75% amino acid sequence identity, still more at least about 80% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, even more preferably at least about 90% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, yet more preferably at least about 95% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, still more preferably at least about 98% amino acid sequence identity, and most preferably at least about 99% to the amino acid sequence shown in SEQ ID NOS:8, 16 or 247, and have comparable or enhanced production activity to the precursor polypeptide.
As indicated above, in some embodiments, the present invention provides vectors comprising the aforementioned polynucleotides. In some preferred embodiments, the vector is an expression vector in which the DNA sequence encoding the protease of the invention is operably linked to additional segments required for transcription of the DNA. In some preferred embodiments, the expression vector is derived from plasmid or viral DNA, or in alternative embodiments, contains elements of both. Exemplary vectors include, but are not limited to pXX, pC194, pJH101, pE194, pHP13 (Harwood and Cutting (eds), Molecular Biological Methods for Bacillus, John Wiley & Sons, [1990], in particular, chapter 3; suitable replicating plasmids for B. subtilis include those listed on page 92; Perego, M. (1993) Integrational Vectors for Genetic Manipulations in Bacillus subtilis, p. 615-624; A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other Gram-positive bacteria: biochemistry, physiology and molecular genetics, American Society for Microbiology, Washington, D.C.).
In some preferred embodiments, the vector pXX finds use in the construction of vectors comprising the polynucleotides described herein (e.g., pXX-049; See,
In some preferred embodiments, the expression vector contains a multiple cloning site cassette which preferably comprises at least one restriction endonuclease site unique to the vector, to facilitate ease of nucleic acid manipulation. In some further preferred embodiments, the vector also comprises one or more selectable markers (e.g., antimicrobial markers such as erythromycin, actinomycin, chloramphenicol, and/or tetracycline). In yet other embodiments, a multicopy replicating plasmid finds use for integration of the plasmid into the Bacilllus genomic DNA, using methods known in the art.
For expression and production of protein(s) of interest e.g. a protease, in a cell, at least one expression vector comprising at least one copy of a polynucleotide encoding the modified protease, and preferably comprising multiple copies, is transformed into the cell under conditions suitable for expression of the protein(s). In some particularly preferred embodiments, the sequences encoding the protein of interest e.g. proteases (as well as other sequences included in the vector) are integrated into the genome of the host cell, while in other embodiments, the plasmids remain as autonomous extra-chromosomal elements within the cell. Thus, the present invention provides both extrachromosomal elements as well as incoming sequences that are integrated into the host cell genome.
Precursor and modified proteases are produced in host cells of any suitable Gram-positive microorganism, including bacteria and fungi. For example, in some embodiments, the modified protease is produced in host cells of fungal and/or bacterial origin. In some embodiments, the host cells are Bacillus sp., Streptomyces sp., Escherichia sp. or Aspergillus sp. In some preferred embodiments, the modified proteases are produced by host cells of the genus Bacillus. Examples of Bacillus host cells that find use in the production of the modified proteins of the present invention include, but are not limited to B. licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B. pumilus, B. thuringiensis, B. clausii, and B. megaterium, as well as other organisms within the genus Bacillus. In some particularly preferred embodiments, B. subtilis host cells find use. U.S. Pat. Nos. 5,264,366 and 4,760,025 (RE 34,606) describe various Bacillus host strains that find use in the present invention, although other suitable strains find use in the present invention.
Industrial strains that find use in the present invention include non-recombinant (i.e., wild-type) Bacillus strains, as well as variants of naturally occurring strain and/or recombinant strains. In some preferred embodiments, the host strain is a recombinant strain, wherein a polynucleotide encoding a polypeptide of interest has been introduced into the host. In some preferred embodiments, the host strain is a B. subtilis host strain and particularly a recombinant Bacillus subtilis host strain. Numerous B. subtilis strains are known, including but not limited to 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 through PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211strain (See e.g., Hoch et al., Genetics, 73:215-228 [1973]) (See also, U.S. Pat. No. 4,450,235; U.S. Pat. No. 4,302,544; and EP 0134048; each of which is incorporated by reference in its entirety). The use of B. subtilis as an expression host well known in the art (See e.g., See, Palva et al., Gene 19:81-87 [1982]; Fahnestock and Fischer, J. Bacteriol., 165:796-804 [1986]; and Wang et al., Gene 69:39-47 [1988]).
In some embodiments, a preferred Bacillus host is a Bacillus sp. that includes a mutation or deletion in at least one of the following genes, degU, degS, degR and degQ. Preferably the mutation is in a degU gene, and more preferably the mutation is degU(Hy)32. (See e.g., Msadek et al., J. Bacteriol., 172:824-834 [1990]; and Olmos et al., Mol. Gen. Genet., 253:562-567 [1997]). A more particularly preferred host strain is a Bacillus subtilis carrying a degU32(Hy) mutation. In some further embodiments, the Bacillus host comprises a mutation or deletion in scoC4, (See, e.g., Caldwell et al., J. Bacteriol., 183:7329-7340 [2001]); spoIIE (See, Arigoni et al., Mol. Microbiol., 31:1407-1415 [1999]); and/or oppA or other genes of the opp operon (See e.g., Perego et al., Mol. Microbiol., 5:173-185 [1991]). Indeed, it is contemplated that any mutation in the opp operon that causes the same phenotype as a mutation in the oppA gene will find use in some embodiments of the altered Bacillus strain of the present invention. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, an altered Bacillus that can be used to produce the modified proteases of the invention is a Bacillus host strain that already includes a mutation in one or more of the above-mentioned genes (in some embodiments, mutations in other genes are also present). In some alternative embodiments, an altered Bacillus further engineered to include mutations of one or more of the above-mentioned genes finds use.
Host cells are transformed with modified polynucleotides encoding the modified proteases of the present invention using any suitable method known in the art. Whether the modified polynucleotide is incorporated into a vector or is used without the presence of plasmid DNA, it is introduced into a microorganism, in some embodiments, preferably an E. coli cell or a competent Bacillus cell. Methods for introducing DNA into Bacillus cells involving plasmid constructs and transformation of plasmids into E. coli are well known. In some embodiments, the plasmids are subsequently isolated from E. coli and transformed into Bacillus. However, it is not essential to use intervening microorganisms such as E. coli, and in some embodiments, a DNA construct or vector is directly introduced into a Bacillus host.
Those of skill in the art are well aware of suitable methods for introducing polynucleotide sequences into Bacillus cells (See e.g., Ferrari et al., “Genetics,” in Harwood et al. (ed.), Bacillus, Plenum Publishing Corp. [1989], pages 57-72; Saunders et al., J. Bacteriol., 157:718-726 [1984]; Hoch et al., J. Bacteriol., 93:1925-1937 [1967]; Mann et al., Current Microbiol., 13:131-135 [1986]; and Holubova, Folia Microbiol., 30:97 [1985]; Chang et al., Mol. Gen. Genet., 168:11-115 [1979]; Vorobjeva et al., FEMS Microbiol. Lett., 7:261-263 [1980]; Smith et al., Appl. Env. Microbiol., 51:634 [1986]; Fisher et al., Arch. Microbiol., 139:213-217 [1981]; and McDonald, J. Gen. Microbiol., 130:203 [1984]). Indeed, such methods as transformation, including protoplast transformation and congression, transduction, and protoplast fusion are known and suited for use in the present invention. Methods of transformation are particularly preferred to introduce a DNA construct provided by the present invention into a host cell.
In addition to commonly used methods, in some embodiments, host cells are directly transformed (i.e., an intermediate cell is not used to amplify, or otherwise process, the DNA construct prior to introduction into the host cell). Introduction of the DNA construct into the host cell includes those physical and chemical methods known in the art to introduce DNA into a host cell without insertion into a plasmid or vector. Such methods include, but are not limited to calcium chloride precipitation, electroporation, naked DNA, liposomes and the like. In additional embodiments, DNA constructs are co-transformed with a plasmid, without being inserted into the plasmid. In further embodiments, a selective marker is deleted from the altered Bacillus strain by methods known in the art (See, Stahl et al., J. Bacteriol., 158:411-418 [1984]; and Palmeros et al., Gene 247:255-264 [2000]).
As indicated above, in some embodiments of the present invention, nucleic acid encoding at least one modified polypeptide of interest is introduced into a host cell via an expression vector capable of replicating within the host cell. Suitable replicating and integrating plasmids for Bacillus known in the art (See e.g., Harwood and Cutting (eds), Molecular Biological Methods for Bacillus, John Wiley & Sons, [1990], in particular, chapter 3; suitable replicating plasmids for B. subtilis include those listed on page 92). Although there are technical hurdles, those of skill in the art know that there are several strategies for the direct cloning of DNA in Bacillus.
Methods known in the art to transform Bacillus, include such methods as plasmid marker rescue transformation, which involves the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (Contente et al., Plasmid 2:555-571 [1979]; Haima et al., Mol. Gen. Genet., 223:185-191 [1990]; Weinrauch et al., J. Bacteriol., 154:1077-1087 [1983]; and Weinrauch et al., J. Bacteriol., 169:1205-1211 [1987]). In this method, the incoming donor plasmid recombines with the homologous region of the resident “helper” plasmid in a process that mimics chromosomal transformation.
Other methods involving transformation by protoplast transformation are known in the art (See e.g., Chang and Cohen, Mol. Gen. Genet., 168:111-115 [1979]; Vorobjeva et al., FEMS Microbiol. Lett., 7:261-263 [1980]; Smith et al., Appl. Env. Microbiol., 51:634 [1986]; Fisher et al., Arch. Microbiol., 139:213-217 [1981]; McDonald [1984] J. Gen. Microbiol., 130:203 [1984]; and Bakhiet et al., 49:577 [1985]). In addition, Mann et al., (Mann et al., Curr. Microbiol., 13:131-135 [1986]) describe transformation of Bacillus protoplasts, and Holubova (Holubova, Microbiol., 30:97 [1985]) describe methods for introducing DNA into protoplasts using DNA-containing liposomes. In some preferred embodiments, marker genes are used in order to indicate whether or not the gene of interest is present in the host cell.
In addition to these methods, in other embodiments, host cells are directly transformed. In “direct transformation,” an intermediate cell is not used to amplify, or otherwise process, the modified polynucleotide prior to introduction into the host (i.e., Bacillus) cell. Introduction of the modified polynucleotide into the host cell includes those physical and chemical methods known in the art to introduce modified polynucleotide into a host cell without insertion into a plasmid or vector. Such methods include but are not limited to the use of competent cells, as well as the use of “artificial means” such as calcium chloride precipitation, electroporation, etc. to introduce DNA into cells. Thus, the present invention finds use with naked DNA, liposomes and the like. In yet other embodiments, the modified polynucleotides are co-transformed with a plasmid without being inserted into the plasmid.
More particularly, the present invention provides constructs, vectors comprising polynucleotides described herein, host cells transformed with such vectors, proteases expressed by such host cells, expression methods and systems for the production of homologous or heterologous serine protease enzymes derived from microorganisms (in particular, members of the genus Bacillus). In some embodiments, the modified polynucleotide(s) encoding modified serine protease(s) are used to produce recombinant host cells suitable for the expression of the modified serine protease(s). In some preferred embodiments, the expression hosts are capable of enhancing the secretion of the mature forms of the modified protease(s) thus increasing the commercial production of proteases.
In some embodiments, the host cells and transformed cells of the present invention are cultured in conventional nutrient media. The suitable specific culture conditions, such as temperature, pH and the like are known to those skilled in the art. In addition, some preferred culture conditions may be found in the scientific literature such as Hopwood (2000) Practical Streptomyces Genetics, John Innes Foundation, Norwich UK; Hardwood et al., (1990) Molecular Biological Methods for Bacillus, John Wiley and from the American Type Culture Collection (ATCC).
In some embodiments, host cells transformed with polynucleotide sequences encoding modified proteases are cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant host cell comprising a modified protease of the present invention is secreted into the culture media. In some embodiments, other recombinant constructions join the heterologous or homologous polynucleotide sequences to nucleotide sequence encoding a protease polypeptide domain which facilitates purification of the soluble proteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53).
Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3:263-281), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego Calif.) between the purification domain and the heterologous protein also find use to facilitate purification.
In some preferred embodiments, the cells transformed with polynucleotide sequences encoding heterologous or homologous protein or endogenously having said protein are cultured under conditions suitable for the expression and recovery of the encoded protein from the cell culture medium. In some embodiments, other recombinant constructions include heterologous or homologous polynucleotide sequences to nucleotide sequence encoding a polypeptide domain which facilitates purification of soluble protein (e.g., tags of various sorts) (Kroll et al., DNA Cell. Biol., 12:441-53 [1993]).
Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath, Prot. Expr. Purif., 3:263-281 [1992]), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and the heterologous protein finds use in facilitating purification.
In some preferred embodiments, the transformed host cells of the present invention are cultured in a suitable nutrient medium under conditions permitting the expression of the present protease, after which the resulting protease is recovered from the culture. The medium used to culture the cells comprises any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection). In some embodiments, the protease produced by the cells is recovered from the culture medium by conventional procedures, including, but not limited to separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt (e.g., ammonium sulfate), chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.). Thus, any method suitable for recovering the protease(s) of the present invention finds use in the present invention. Indeed, it is not intended that the present invention be limited to any particular purification method.
As indicated above, the polypeptides of the invention are produced as mature enzymes at levels greater than the mature enzymes processes from their corresponding unmodified precursor polypeptides. In preferred embodiments of the present invention, the mutations within the pro region of a precursor polypeptide enhance the secretion/expression of the mature polypeptide when compared to a corresponding mature protease that has been processed from a precursor protease when produced by a Bacillus strain under the same conditions.
One measure of enhancement can be determined as an activity ratio, which can be expressed as the ratio of the enzymatic activity of the mature form processed from the modified protease to the enzymatic activity of the mature form processed from the precursor protease. A ratio equal or greater than 1 indicates that the mature form of modified protease is produced at levels equal or greater than those at which the mature form of precursor protease is produced. For example, an activity ratio of 1.5 indicates that the mature protease that has been processed from a modified protease is produced at 1.5 times the level at which the mature protease that is processed from the precursor protease i.e. the modified protease yields 50% more mature protease than the unmodified precursor protease. In some embodiments, the activity ratio is at least 1, at least about 1.05, about at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8. at least about 1.9, and at least about 2. In other embodiments, the activity ratio is at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9 and at least about 3. In yet other embodiments, the activity ratio is at least about 3.5, at least about 4.0, and at least about 5. Thus, in some embodiments, production of the mature protease processed from the modified protease is enhanced by at least about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 4.0%, about 5.0%, about 8.0%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more, compared to the corresponding mature protease processed from the unmodified precursor protease. In other embodiments, production of the mature form of the protease that is processed from the modified protease is enhanced by at least about 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, and up to at least about 200%, or more compared to the corresponding production of the mature form of the protease that was processed from the unmodified precursor protease. In some embodiments, the enhanced production of the modified protease is determined based on the ratio of the proteolytic activity of the mature form processed from the modified protease compared to the proteolytic activity of the mature form of the corresponding unmodified precursor protease.
Other means for determining the levels of secretion of a heterologous or homologous protein in a host cell and detecting secreted proteins include, but are not limited to methods that use either polyclonal or monoclonal antibodies specific for the protein. Examples include, but are not limited to enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See e.g., Maddox et al., J. Exp. Med., 158:1211 [1983]). In some preferred embodiments of the present invention, secretion is higher using the methods and compositions provided herein than when using the same methods or compositions, but where a peptide transport protein or gene product of a peptide transport operon has not been introduced.
There are various assays known to those of ordinary skill in the art for detecting and measuring activity of polypeptides of the invention. In particular, assays are available for measuring protease activity that are based on the release of acid-soluble peptides from casein or hemoglobin, measured as absorbance at 280 nm or colorimetrically using the Folin method (See e.g., Bergmeyer et al., “Methods of Enzymatic Analysis” vol. 5, Peptidases, Proteinases and their Inhibitors, Verlag Chemie, Weinheim [1984]). Some other assays involve the solubilization of chromogenic substrates (See e.g., Ward, “Proteinases,” in Fogarty (ed.)., Microbial Enzymes and Biotechnology, Applied Science, London, [1983], pp 251-317). Other exemplary assays include, but are not limited to succinyl-Ala-Ala-Pro-Phe-para nitroanilide assay (SAAPFpNA) and the 2,4,6-trinitrobenzene sulfonate sodium salt assay (TNBS assay). Numerous additional references known to those in the art provide suitable methods (See e.g., Wells et al., Nucleic Acids Res. 11:7911-7925 [1983]; Christianson et al., Anal. Biochem., 223:119-129 [1994]; and Hsia et al., Anal Biochem., 242:221-227 [1999]). It is not intended that the present invention be limited to any particular assay method(s).
In some embodiments, the production of the modified protease by a microorganism is determined by using a ratio of the activity of a mature protease processed from a modified precursor protease compared to the activity of the mature protease processed from an unmodified precursor protease. In some particularly preferred embodiments, ratio of 1 or greater is desired.
Other means for determining the levels of production of a protein of interest e.g. a protease, in a host cell and detecting expressed proteins include the use of immunoassays with either polyclonal or monoclonal antibodies specific for the protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and fluorescent activated cell sorting (FACS). However, other methods are known to those in the art and find use in assessing the protein of interest (See e.g., Hampton et al., Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn. [1990]; and Maddox et al., J. Exp. Med., 158:1211 [1983]). In some preferred embodiments, secretion of a protein of interest is higher in the altered strain obtained using the present invention than in a corresponding unaltered host. As known in the art, the altered Bacillus cells produced using the present invention are maintained and grown under conditions suitable for the expression and recovery of a polypeptide of interest from cell culture (See e.g., Hardwood and Cutting (eds.) Molecular Biological Methods for Bacillus, John Wiley & Sons [1990]). It is not intended that the present invention be limited to any particular assay method(s).
All publications and patents mentioned herein are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art and/or related fields are intended to be within the scope of the present invention.
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND (not done); NA (not applicable); rpm (revolutions per minute); H2O (water); dH2O (deionized water); (HCl (hydrochloric acid); aa (amino acid); by (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl2 (magnesium chloride); NaCl (sodium chloride); w/v (weight to volume); v/v (volume to volume); g (gravity); OD (optical density); Dulbecco's phosphate buffered solution (DPBS); OD280 (optical density at 280 nm); OD600 (optical density at 600 nm); A405 (absorbance at 405 nm); PAGE (polyacrylamide gel electrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PBST (PBS+0.25% TWEEN®-20); PEG (polyethylene glycol); PCR (polymerase chain reaction); SDS (sodium dodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); HEPES (N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPES buffered saline); SDS (sodium dodecylsulfate); bME, BME and βME (beta-mercaptoethanol or 2-mercaptoethanol); Tris-HCl (tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine (N-[tris-(hydroxymethyl)-methyl]-glycine); DMSO (dimethyl sulfoxide); Taq (Thermus aquaticus DNA polymerase); Klenow (DNA polymerase I large (Klenow) fragment); rpm (revolutions per minute); EGTA (ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid); EDTA (ethylenediaminetetracetic acid); bla (β-lactamase or ampicillin-resistance gene); DNA2.0 (DNA2.0, Menlo Park, Calif.); OXOID (Oxoid, Basingstoke, Hampshire, UK); Corning (Corning Life Sciences, Corning, N.Y.); ATCC (American Type Culture Collection, Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Pisacataway, N.J.); NCBI (National Center for Biotechnology Information); Applied Biosystems (Applied Biosystems, Foster City, Calif.); Clontech (CLONTECH Laboratories, Palo Alto, Calif.); Operon Technologies (Operon Technologies, Inc., Alameda, Calif.); Bachem (Bachem Bioscience, Inc., King of Prussia, Pa.); Difco (Difco Laboratories, Detroit, Mich.); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Millipore (Millipore, Billerica, Mass.); Bio-Rad (Bio-Rad, Hercules, Calif.); Invitrogen (Invitrogen Corp., San Diego, Calif.); NEB (New England Biolabs, Beverly, Mass.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pierce (Pierce Biotechnology, Rockford, Ill.); Takara (Takara Bio Inc. Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science (EM Science, Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia, Calif.); Molecular Devices (Molecular Devices, Corp., Sunnyvale, Calif.); R&D Systems (R&D Systems, Minneapolis, Minn.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); and Microsoft (Microsoft, Inc., Redmond, Wash.).
Site-saturation mutagenesis of the pro sequence of the Bacillus clausii alkaline protease V049 precursor protease was performed using the QuikChange® site-directed mutagenesis kit (QC; Stratagene) according to the directions provided by the manufacturer. Site-saturation libraries were created to include modified polynucleotides sequences of the precursor polynucleotide encoding the B. clausii protease variant V049. The sequence encoding the pro region of the V049 polynucleotide contained in the pXX-V049 plasmid (
*The primer names provided reflect the amino acid position of the substitution; “R” indicates that the primer is the reverse primer and “F” indicates that the primer is a forward primer. For example, V049-108F is the forward primer that was used in the substitution of amino acid at position 108 of the V049 precursor protease.
**“Bases left” and ***“Bases Right” indicate the number of bases to the left and to the right of the mutating codon that are present in the primer. These bases are complementary to the bases of the template precursor polynucleotide bases (i.e. V049).
The QC reaction consisted of 40.25 μL of sterile distilled H2O, 5 μL of PfuTurbo 10× buffer from the kit, 1 μL dNTPs from the kit, 1.25 μL of forward primer (100 ng/μL), 1.25 μL reverse primer (100 ng/μL), 0.25 μL of pMSAT-NcoI miniprep DNA as template (˜50 ng), and 1 μL of PfuTurbo from the kit, for a total of 50 μL. The cycling conditions were 95° C. for 1 min, once, followed by 19-20 cycles of 95° C. for 1 min., 55° C. for 1 min, and 68° C. for 12 min. To analyze the reaction, 5 μL of the reaction was run on a 1.2% E-gel (Invitrogen) upon completion. Next, DpnI digestion was carried out twice sequentially, with 1 μL and 1 μL of enzyme at 37° C. for 2 to 8 hours. A negative control was carried out under similar conditions, but without any primers. Then, 1 μL of the DpnI-digested reaction product was transformed into 50 μL of one-shot TOP10 electrocompetent cells (Invitrogen) using a BioRad electroporator. Then, 1 ml of SOC provided with the TOP10 cells (Invitrogen) were added to the electroporated cells and incubated with shaking for 1 hour before plating on LA plates containing 5 ppm chloramphenicol. The plates were incubated at 37° C. overnight. After this incubation, 96 colonies from each of the libraries (i.e., each site) were inoculated in 200 μL of LB containing 10-50 ppm of Chloramphenicol in 96-well microtiter plates and grow overnight at 37° C. The plates were frozen at −80° C. after addition of glycerol to 20% final concentration the next day, and they were used for high throughput sequencing with the V049SEQ-R2 primers.
Similarly, mutations of codons encoding two or more amino acids of the pro region of the precursor protease V049 are performed using the QuikChange® Multi Site-Directed mutagenesis (QCMS; Stratagene). The QCMS reaction is performed using 19.25 μL of sterile distilled H2O, 2.5 μL of 10× buffer from the kit, 1 μL dNTPs from the kit, 1 μL of 5′ phosphorylated forward primer (100 ng/μL), 0.25 μL of pMSAT-NcoI miniprep DNA as template (˜50 ng), and 1 μL of the enzyme blend from the kit for a total of 25 μL. The cycling conditions are 95° C. for 1 min once, followed by 30 cycles of 95° C. for 1 min, 55° C. for 1 min, and 65° C. for 12 min. To analyze the reaction product, 2.5 μL of the reaction are run on a 1.2% E-gel (Invitrogen) upon completion. Next, DpnI digestion is carried out twice sequentially, with 1 ul and then 0.5 μL of enzyme at 37° C. for 2 to 8 hours. The controls, transformation, and sequencing are performed as for the QC method described above.
Plasmids pXX-049 containing polynucleotides encoding the modified proteins of interest were digested twice with DpnI for 3-5 hours at 37 C.
Transformation and Screening in E. coli
1 ul of DpnI digested plasmid DNA was used to transform E. coli Top10 cells by electroporation. The transformed cells were plated onto LB agar plates containing 5 ppm CMP (chloramphenicol), and colonies were allowed to grow overnight. 96 individual colonies were picked and transferred to corresponding wells of a 96-well micro-titer plate containing LB+5 ppm CMP+50 ppm carbenicillin. Cultures were grown overnight at 37° C. while shaking at 250 rpm. Glycerol stock was added in the culture to a final concentration of 10%. Plasmid DNA was prepared from the 96 E. coli cultures, and a portion of the plasmid DNA preparation was sequenced (Cogenics, Morrisville, N.C.). Automated sequence analysis was performed using Phrep, Phrap, Consed, Custal W software.
Transformation into Bacillus subtilis
A second portion of the plasmid DNA was used to transform B. subtilis host cells. Two microliters of plasmid DNA from each of the 96 E. coli cultures carrying the appropriate mutations were used to transform 100 ul of B. subtilis ComK competent cells. The cells were incubated at 37° C. for 45 minutes while shaking at 250 rpm. Cells from the 96 transformation mixture were plated onto LA containing 1.6% skim milk and 5 ppm CMP and incubated overnight in at 37° C. incubator.
Four colonies, from each of the 96 transformations, were picked and individually transferred to a micro-titer plate containing 150 ul of LB and 5 ppm CMP per well. A number of wells of the micro-titer plate contained the appropriate controls. The micro-titer plates were then incubated for four hours at 37° C. while rotating at 250 rpm. 10 ul of each of the cultures were transferred to a new micro-titer plate containing 140 ul of Grant II media+5 ppm CMP, pH 7.3. GrantII media was prepared as follows: Solution I: 10 g of Soytone were dissolved in 500 ml water and autoclaved for 20-25 minutes; Solution II: 3 ml of 1M K2HPO4, 75 g glucose, 3.6 g urea, 100 ml Grant's 10× MOPS were diluted into 400 ml water. Solutions I and II were mixed and the pH adjusted to pH7.3 with HCl/NaOH. The final volume was adjusted to 1 L, and the final solution was sterilized through 0.22-um PES filter.
The micro-titer plate cultures were incubated in shaker at 37° C., 250 rpm. Samples were taken at regular intervals (up to 40 hours) for assay analysis.
Each of the B. subtilis cultures obtained as described in Example 2, was assayed for the production of the modified proteases. The enzymes produced were assayed for activity against the substrate, succinyl -L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanalide (AAPF). The assay measured the production of modified protease as the increase in absorbance at 405 nm/min resulting from the hydrolysis and release of p-nitroanaline (Estell et al., J Biol. Chem., 260:6518-6521 (1985)). The measurements were made using the Sofmax Pro software, and the specified conditions were set as: Type: Kinetic; Reduction: Vmax Points (Read best 15/28 points); Lm1: 405 nm; Time: 5 minutes; and Interval: 11 Seconds. Ten microliters of each of the B. subtilis cultures were diluted 100 ul of Tris Buffer, containing 10 mM Tris+0.005% TWEEN®-80, pH 8.6; and 25 ul of 100 mg/ml AAPF. The relative activity of each of the modified proteases was calculated, and the effect of each amino acid substitution on the production of the corresponding modified protease was determined as a ratio of the activity of the mature protease processed from each modified protease to the activity of the mature protease processed from the unmodified V049 precursor protease. The results are given in Table 2.
Once the DNA construct was stably integrated into a competent Bacillus subtilis strain, the activity of the modified proteases was measured in microtiter assays and the activity was compared to the activity of the corresponding precursor protease.
Ten microliters of overnight Grant II Media cell cultures were diluted to 100 ul of Tris Buffer, containing 10 mM Tris+0.005% TWEEN®-80 pH 8.6; and 25 ul of 100 mg/ml AAPF substrate were used to assay for protease activity. Assays were done in microtiter plates and the Softmax Pro Software was used.
The results showed that amino acid substitution of most of the amino acids of the precursor V049 protease lead to an enhanced production of the mature form of the protease. In addition, site saturation of each of the substituted amino acids showed that each amino acid can be substituted by two or more amino acids at the same position to increase the production of the mature form relative to that obtained from the precursor protease having unmodified pro region.
In this example, the pro region of the subtilisin from Bacillus lentus, called GG36 (SEQ ID NO:244), was mutagenized at position 33 and position 59 using the same experimental protocol described in the examples above. The precursor polynucleotide sequence (SEQ ID NO:240) was mutated, a construct was made, and the modifications were tested for increasing the protease expression as described above.
The results showed that amino acid substitutions E33G, E33Q and E33V all increased the production of the wild-type mature GG36 protease by at least 50% above that of the unmodified precursor. The mutation H32K increased the production of the wild-type mature protease by 20%.
These results show that mutations in the pro region of a full-length protease can improve the expression not only of variant precursor proteins, but also of naturally-occurring enzymes.
This application is a divisional of U.S. patent application Ser. No. 12/047,157, filed Mar. 12, 2008, and claims priority to U.S. provisional application Ser. No. 60/906,734, filed Mar. 12, 2007, both of which are herein incorporated by reference in their entirety.
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| English translation of Office Action dated Apr. 7, 2015 in a Japanese counterpart application, Application No. 2009-553618. |
| Number | Date | Country | |
|---|---|---|---|
| 20140295526 A1 | Oct 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60906734 | Mar 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12047157 | Mar 2008 | US |
| Child | 14305884 | US |
