Patent Application
20160230156
Nucleases are useful reagents for removing unwanted nucleic acids from protein preparations. Descriptions of certain nucleases and their preparation by cloning are provided by Eaves et al. (J. Bacteriol. 85:273-8 (1963)), Filimonova et al. (Biochem. Mol. Biol. Int. 33(6):1229-36 (1994)), Ball et. al. (Gene 57(2-3):183-92 (1987), Molin et. al. (U.S. Pat. No. 5,173,4185), and Friedhoff et al. (Protein Expr. Purif. 5(1):37-43 (1994)).
Exonuclease I (Exo I) digests single-stranded DNA in a 3′ to 5′direction producing 5′ mononucleotides. This enzyme is particularly useful in preparing amplified DNA products, such as PCR products, for sequencing. It degrades residual primers from the amplification reaction that would otherwise be carried over into the sequencing reaction. U.S. Pat. Nos. 5,741,676 and 5,756,285 generally disclose methods for DNA sequencing via amplification, both of which are hereby incorporated herein by reference. (See also R. L. Olsen et al., Comp. Biochem. Physiol., vol. 99B, No. 4, pp. 755-761 (1991)).
Amplification primers carried over into a sequencing reaction could act as sequencing primers and generate sequencing reaction products, thereby creating a background of secondary sequences which would obscure or interfere with observing the desired sequence. Both the concentration and specific activity (purity) of commercially available Exonuclease I may vary over a wide range. Commonly the enzyme is manufactured to a specific activity between 50,000 and 150,000 units of enzyme per mg and supplied for the purpose of processing amplified DNA at a concentration around 10 units per microliter. Enzyme with either higher or lower specific activity and either more or less concentrated could be employed in the described applications by suitable alterations in the applied protocol, such as adding less or more volume (or amount) of enzyme, respectively.
Alkaline Phosphatases, as exemplified by Shrimp Alkaline Phosphatase (SAP) and Calf Intestinal Alkaline Phosphatase (CIP), catalyze the hydrolysis of 5′-phosphate residues from DNA, RNA, and ribo- and deoxyribonucleoside triphosphates (dNTPs or nucleotide triphosphates). SAP is particularly useful in preparing amplified products, such as PCR products, for sequencing because it can readily be inactivated by heat prior to performing a sequencing reaction. SAP degrades residual dNTPs from the amplification reaction. If residual dNTPs are carried over from the amplification reaction to the sequencing reaction, they add to, and thereby alter, the concentration of dNTPs in the sequencing reaction in an indeterminant and non-reproducible fashion. Since, within narrow limits, high quality sequencing requires specific ratios between the sequencing reaction dNTPs and ddNTPs, an alteration in the concentration of dNTPs may result in faint sequencing reaction signals.
Prior to sequencing or other analyses, Exo I and SAP are frequently used to process PCR reaction products. There is a need in the art for an improved composition that provides both nuclease and phosphatase activity.
Disclosed herein is a novel recombinant fusion protein. This fusion protein possesses two enzymatic activities, a nuclease activity and a phosphatase activity. Methods for the isolation and use of this novel protein are also disclosed.
In some embodiments are provided a recombinant fusion protein. “Recombinant” or “recombinant nucleic acid” or “recombinant gene” or “recombinant DNA molecule” or “recombinant nucleic acid sequence” indicates that the nucleotide sequence or arrangement of its parts is not a native configuration, and has been manipulated by molecular biological techniques. The term implies that the DNA molecule is comprised of segments of DNA that have been artificially joined together, for example, the polynucleotide encoding a nuclease and a phosphatase activity disclosed herein. Protocols and reagents to manipulate nucleic acids are common and routine in the art (See e.g., Maniatis et al. (eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, 1982; Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, NY, 1989; and Ausubel et al. (eds.), Current Protocols in Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., New York 1994; all of which are herein incorporated by reference).
“Fusion protein” refers to a polypeptide composed of a plurality of components, unjoined in their native state but are joined to form a single continuous polypeptide.
Similarly, a “recombinant protein” or “recombinant polypeptide” refers to a protein molecule that is expressed from a recombinant DNA molecule. Use of these terms indicates that the primary amino acid sequence, arrangement of its domains or nucleic acid elements which control its expression are not native, and have been manipulated by molecular biology techniques. As indicated above, techniques to manipulate recombinant proteins are also common and routine in the art.
As used herein, the word “protein” refers to a full-length protein, a portion of a protein, or a peptide. Proteins can be produced via fragmentation of larger proteins, or chemically synthesized. Proteins may, for example, be prepared by recombinant overexpression in a species such as, but not limited to, bacteria, yeast, insect cells, and mammalian cells. Proteins to be placed in a protein microarray of the invention, may be, for example, are fusion proteins, for example with at least one affinity tag to aid in purification and/or immobilization. In certain aspects of the invention, at least 2 tags are present on the protein, one of which can be used to aid in purification and the other can be used to aid in immobilization. In certain illustrative aspects, the tag is a His tag, a GST tag, or a biotin tag. Where the tag is a biotin tag, the tag can be associated with a protein in vitro or in vivo using commercially available reagents (Invitrogen, Carlsbad, Calif.). In aspects where the tag is associated with the protein in vitro, a Bioease tag can be used (Invitrogen, Carlsbad, Calif.).
As used herein, the term “peptide,” “oligopeptide,” and “polypeptide” are used interchangeably with protein herein and refer to a sequence of contiguous amino acids linked by peptide bonds. As used herein, the term “protein” refers to a polypeptide that can also include post-translational modifications that include the modification of amino acids of the protein and may include the addition of chemical groups or biomolecules that are not amino acid-based. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms “polypeptide,” “peptide” and “protein” include glycoproteins, as well as non-glycoproteins.
In some embodiments are provided a recombinant fusion protein, wherein the recombinant fusion protein possesses two enzymatic activities, wherein the first enzymatic activity is a nuclease activity.
“Nuclease” refers to an enzyme capable of cleaving phosphodiester bonds between the nucleotide subunits of nucleic acids.
In some embodiments are provided a recombinant fusion protein, wherein the recombinant fusion protein possesses two enzymatic activities, wherein the first enzymatic activity is a nuclease activity and the nuclease activity is exonuclease activity.
“Exonuclease” refers to an enzyme that cleaves nucleotides one at a time from the end of a polynucleotide chain via a hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or 5′ end. The “exonuclease” can be a 3′ to 5′ exonuclease or a 5′ to 3′ exonuclease. E. coli exonuclease I and exonuclease III are two commonly used 3′-exonucleases that have 3′-exonucleolytic single-strand degradation activity. E. coli exonuclease VII and T7-exonuclease Gene 6 are two commonly used 5′-3′ exonucleases that have 5′-exonucleolytic single-strand degradation activity.
Exonucleases can be originated from prokaryotes, such as E. coli exonucleases, or eukaryotes, such as yeast, worm, murine, or human exonucleases. Examples of exonucleases that can be used in the disclosed fusion protein include, but are not limited to, E. coli exonuclease I, E. coli exonuclease III, E. coli exonuclease VII, bacteriophage lambda exonuclease, and bacteriophage T7-exonuclease Gene 6, or a combination thereof.
In some embodiments are provided a recombinant fusion protein, wherein the recombinant fusion protein possesses two enzymatic activities, wherein the first enzymatic activity is a nuclease activity and the second enzymatic activity is a phosphatase activity.
“Phosphatase” or “alkaline phosphatase” refers to an enzyme capable of hydrolyzing phosphoric monoesters to produce inorganic phosphoric acids. Alkaline phosphatases are generally known to be metal-dependent enzymes that have low substrate specificity and require metal ions such as magnesium ions (Mg2+) or zinc ions (Zn2+) for enzymatic reactions. Typical alkaline phosphatases include bacterial alkaline phosphatase (BAP), calf intestinal alkaline phosphatase (CIAP), shrimp alkaline phosphatase (SAP) and the like.
In some embodiments are provided a nucleic acid encoding a recombinant fusion protein, wherein the recombinant fusion protein possesses two enzymatic activities, wherein the first enzymatic activity is a nuclease activity and the second enzymatic activity is a phosphatase activity.
“Nucleic acid” refers to polymers of single or double stranded nucleotide. A nucleic acid typically refers to a polynucleotide molecule comprised of a linear strand of two or more nucleotides (deoxyribonucleotides and/or ribonucleotides) or variants, derivatives and/or analogs thereof
In some embodiments the nucleic acid encompasses SEQ ID NO:1.
In some embodiments the recombinant fusion protein possessing both nuclease and phosphatase activity encompasses the polypeptide sequence of SEQ ID NO:2.
In some embodiments are provided a vector with a nucleic acid insert, wherein the nucleic acid insert encodes a recombinant fusion protein, wherein the recombinant fusion protein possesses two enzymatic activities, wherein the first enzymatic activity is a nuclease activity and the second enzymatic activity is a phosphatase activity. In other embodiments, the nucleic acid insert encompasses SEQ ID NO:1. In some embodiments, the nucleic acid encodes the polypeptide of SEQ ID NO:2.
“Vector” refers to any DNA or RNA molecule that acts as an intermediate carrier into which a DNA or RNA segment is inserted for introduction into a host cell for amplification. Such intermediate carriers include plasmids, cosmids, bacteriophages and transposons.
“Host cell” refers to any cell type which is susceptible to transformation, transfection, and/or transduction with a nucleic acid construct. A host cell can be a prokaryotic or eukaryotic cell.
In some embodiments, the recombinant fusion protein is purified.
The terms “purified” and “isolated” as used herein, are synonymous, and refer to a material that is substantially or essentially free from other components. For example, in one embodiment, a recombinant protein is isolated or purified when it is free from other components used in the cloning reaction, or solid state synthesis, isolation or purity is generally determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis, mass spectrometry, or high performance liquid chromatography (HPLC). In one embodiment, a polynucleotide, protein or peptide of the present invention is considered to be isolated when it is the predominant species present in a preparation. A purified protein, peptide or nucleic acid molecule of the invention represents greater than about 80% of the macromolecular species present, greater than about 90% of the macromolecular species present, greater than about 95% of the macromolecular species present, greater than about 96% of the macromolecular species present, greater than about 97% of the macromolecular species present, greater than about 98% of the macromolecular species present, greater than about 99% of the macromolecular species present in a preparation.
In some embodiments are provided a kit encompassing a recombinant fusion protein, wherein the recombinant fusion protein possesses two enzymatic activities, wherein the first enzymatic activity is a nuclease activity and the second enzymatic activity is a phosphatase activity.
The kits of the present invention may also comprise instructions for performing one or more methods described herein and/or a description of one or more compositions or reagents described herein. Instructions and/or descriptions may be in printed form and may be included in a kit insert. A kit also may include a written description of an internet location that provides such instructions or descriptions.
At the outset, several chimeric fusion proteins possessing both nuclease and phosphatase activity were envisioned and tested. For instance, the combination of exonuclease and shrimp alkaline phosphatase activity of SEQ ID NO. 3 and SEQ ID NO. 4 was constructed and tested. This chimeric fusion protein failed in early stages of development for lacking solubility and desired enzymatic activity. A number of other chimeric exonuclease/phosphatase fusion proteins were also tested but failed.
Purification of Chimeric Enzyme from Bacterial Host Cells
In order to isolate the fusion protein (SEQ ID NO. 2) the following procedure was used, schematically represented in
To precipitate the chimeric exonuclease/phosphatase fusion protein, after centrifugation 40% saturated ammonium sulfate was added to the supernatant and then this was subjected to centrifugation. The resulting pellet is resuspended in 25 mM HEPES (pH 6.3).
The resuspended pellet was then loaded onto a POROS® HS cation exchange column equilibrated with 25 mM HEPES (pH 6.3). Peak fractions from a 0-1 M NaCl gradient were pooled and diluted 1:2 prior to loading onto a POROS® HQ anion exchange column equilibrated with 25 mM HEPES (pH 6.3). Peak fractions were pooled and dialyzed against 25 mM HEPES (pH 8.2), 2 mM CaCl2 and 50% glycerol.
This methodology resulted in approximately 3 mg. of the expressed chimeric exonuclease/phosphatase fusion protein per gram of bacteria.
At a number of steps in the purification process, samples were taken and analyzed by SDS-PAGE. Results of these analyses are depicted in
The chimeric exonuclease/phosphatase fusion protein (SEQ ID NO. 2) was utilized in a DNA sequencing workflow to assess its functional characteristics. Human genomic DNA was used as template for PCR amplification of amplification of 639 bp portion of the HLA locus. After the completion of thermal cycling, various molar concentrations of the chimeric exonuclease/phosphatase fusion protein were added to the PCR amplification tubes and the reactions were incubated for 15 minutes at 37° C., followed by 15 minutes at 80° C. ExoSAP-IT® was added to some amplification tubes as a comparative control.
After the incubation was complete, each reaction was subjected to DNA cycle sequencing. Resulting QV and electropherograms from the cycling sequencing are depicted in
In addition to the human HLA locus DNA sequencing was performed using a bacterial 16S ribosomal RNA gene (rDNA) sequence as a template. To accomplish this, an approximately 500 bp amplicon was generated by PCR amplification of a bacterial rDNA sequence. After PCR amplification, the chimeric exonuclease/phosphatase fusion protein was added to the reaction and incubated, followed by cycle sequencing and gel electrophoresis. Results from representative experiments using the protein of SEQ ID NO. 2 are depicted in
This application is a U.S. National Application filed under 35 U.S.C. 371 of PCT Application Serial No. PCT/US2014/030515 filed Mar. 17, 2014, which claims a priority benefit under 35 U.S.C. §119(e) from U.S. Provisional Application No. 61/798,671 filed Mar. 15, 2013, which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/30515 | 3/17/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61798671 | Mar 2013 | US |
