Patent Application
20220275028
This invention relates to recombinant cell-free expression systems and methods of using the same for high yield in vitro production of biological materials.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 5, 2022, is named 90125-00097-Sequence-Listing_Amended.txt and is 427 Kbytes in size
Cell-free expression systems (also known as in vitro transcription/translation, cell-free protein expression, cell-free translation, or cell-free biosynthesis) represent a molecular biology technique that enables researchers to express functional proteins or other target molecules in vitro. Such systems enable in vitro expression of proteins or other small molecules that are difficult to produce in vivo, as well as high-throughput production of protein libraries for protein evolution, functional genomics, and structural studies. Another advantage of such systems is that often the target protein to be expressed may be toxic to a host cell, or generally incompatible with cellular expression, making in vivo systems impractical if not wholly ineffective vehicles for protein expression. Compared to in vivo techniques based on bacterial or tissue culture cells, in vitro protein expression is considerably faster because it does not require gene transfection, cell culture or extensive protein purification.
More specifically, cell-free expression systems generate target molecules and complexes such as RNA species and proteins without using living cells. A typical cell-free expression system may utilize the biological components/machinery found in cellular lysates to generate target molecules from DNA containing one or more target genes. Common components of a typical cell-free expression system reaction may include a cell extract generally derived from a cell culture lysate, an energy source such as ATP, a supply of amino acids, cofactors such as magnesium, and the nucleic acid synthesis template with the desired genes, typically in the form of a plasmid synthesis template, or linear expression (or synthesis) template (LET or LST). A cell extract may be obtained by lysing the cell of interest and removing the cell walls, genomic DNA, and other debris through centrifugation or other precipitation methods. The remaining portions of the lysate or cell extract may contain the necessary cell machinery needed to express the target molecule.
A common cell-free expression system involves cell-free protein synthesis (CFPS). To produce one or more proteins of interest, typical CFPS systems harness an ensemble of catalytic components necessary for energy generation and protein synthesis from crude lysates of microbial, plant, or animal cells. Crude lysates contain the necessary elements for DNA to RNA transcription, RNA to protein translation, protein folding, and energy metabolism (e.g., ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, ribosome release factors, nucleotide recycling enzymes, metabolic enzymes, chaperones, foldases, etc.). Common cell extracts in use today are made from Escherichia coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), and insect cells (ICE), and even mammalian cells (MC).
Cell-free expression systems offer several advantages over conventional in vivo protein expression methods. Cell-free systems can direct most, if not all, of the metabolic resources of the cell towards the exclusive production of one protein. Moreover, the lack of a cell wall and membrane components in vitro is advantageous since it allows for control of the synthesis environment. For example, tRNA levels can be changed to reflect the codon usage of genes being expressed. The redox potential, pH, or ionic strength can also be altered with greater flexibility than in vivo since there is less concerned about cell growth or viability. Furthermore, direct recovery of purified, properly folded protein products can be easily achieved.
Despite many advantageous aspects of cell-free expression systems, several obstacles have previously limited their use as a protein production technology. These obstacles, which are especially present in the E. coli extract-based cell-free systems identified in U.S. Pat. No. 7,118,883, and the yeast extract-based cell-free systems identified in U.S. Pat. No. 9,528,137, include short reaction durations of active protein synthesis, low protein production rates, small reaction scales, a limited ability to correctly fold proteins containing multiple disulfide bonds, and its initial development as a “black-box” science. As a result, there exists a need for an economically viable commercial cell-free expression system that exhibits increased product yield, enhanced component stability, improved protein production rate, and extended reaction time.
As noted above, cell-free systems are not widely used for manufacturing of biologics because of their lack in consistency, yield and possibility to scale. The present inventors previously reported an extract-based cell-free system utilizing exemplary thermophiles to improve the application of such systems by replacing the E. coli machinery with thermostable proteins which led to improved production rates and higher yields, but also including a novel energy regeneration system. (Such novel energy regeneration systems being generally described in PCT Application No. PCT/US201 8/012121, the description, figures, examples, sequences and claims being incorporated herein by reference in their entirety.)
As detailed below, the present inventors have developed a fully recombinant in vitro transcription/translation system, which in some embodiments, incorporate peptide-based components from various exemplary thermophilic bacteria. As noted above, current commercially available cell-free systems are either based on adding necessary transcription/translation machinery from E. coli cell extracts or are based on recombinant E. coli enzymes. Various other sources for extracts have been reported including the use of thermophiles to improve in vitro protein production, but a fully recombinant expression system, including a fully-recombinant expression system based on thermophilic proteins has not been reported until now.
As will be discussed in more detail below, the current inventive technology overcomes the limitations of traditional cell-free expression systems while meeting the objectives of a truly energetically efficient and robust in vitro cell-free expression system that results in longer reaction durations and higher product yields. Specifically, the present invention includes a cell-free system based on thermophiles by recombinantly expressing each protein necessary for transcription/translation and thus enabling continuous flow with better control and fine tuning of the system without encountering huge variables as observed in extract-based batch systems. This system may be useful for small scale protein production in initial research applications as well as for mid-scale applications, such as small animal studies. The current invention allows for large scale manufacturing with the continuous flow approach in novel bioreactors described herein and can replace current manufacturing facilities with much larger footprints and personnel requirements.
One aim of the current invention relates to a recombinant cell-free expression system, the reaction mixture containing all the cell-free reaction components necessary for the in vitro transcription/translation mechanism, amino acids, nucleotides, metabolic components which provide energy, and which are necessary for protein synthesis. In a preferred embodiment, the enzymes identified herein may be sourced from different thermophile bacteria, as opposed to traditional cell-free systems that source components from E. coli or other eukaryotic systems, such as yeast. This thermophilic sourcing strategy provides higher stability during all steps during in vitro translation (tRNA loading, ribosomal peptide biosynthesis), as well as allows for improved performance and longer run-time of the recombinant expression system.
This present inventor's thermophilic sourcing strategy allows for the generation of a recombinant cell-free expression system that exhibits less sensitivity to variations in pH and salt concentrations and may be less affected by increasing phosphate concentration due to ATP hydrolysis. Another benefit of this thermophilic sourcing strategy is that it allows the inventive recombinant cell-free expression system to employ different sets of tRNAs, which are recognized by the thermophilic aminoacyl-tRNA synthetase enzymes, thus enabling full codon coverage for the first time in a cell-free system.
Another aim of the current invention may include a recombinant cell-free expression system, the reaction mixture containing all the cell-free reaction components necessary for the in vitro biosynthesis of biological compounds, proteins, enzymes, biosimilars or chemical modification of small molecules.
Another aim of the current invention may include methods, systems and apparatus for a continuous flow bioreactor system for in vitro transcription, in vitro translation and in vitro biosynthesis of vaccines, biologicals, proteins, enzymes, biosimilars and biosynthesis or chemical modification of small molecules using enzymes in a continuous flow operation.
Another aim of the invention may include one or more isolated nucleotide coding sequences that may form part of a recombinant cell-free expression reaction mixture. In a preferred embodiment, one or more nucleotide coding sequences may be from a thermophilic or other bacteria. In a preferred embodiment, a nucleotide coding sequences may include, but not be limited to: initiation factor nucleotide coding sequences, elongation factor nucleotide coding sequences, release factor nucleotide coding sequences, ribosome-recycling factor nucleotide coding sequences, aminoacyl-tRNA synthetase nucleotide coding sequences, and methionyl-tRNA transformylase nucleotide coding sequences. Additional nucleotide coding sequences may include RNA polymerase nucleotide coding sequences, as well as nucleotide coding sequences identified in the incorporated reference PCT Application No. PCT/US201 8/012121 (the “'121 Application”) related to the inorganic polyphosphate energy-regeneration system incorporated herein.
Another aim of the invention may include the generation of expression vectors having one or more isolated nucleotide coding sequences operably linked to promotor sequence(s) that may be used to transform a bacterial cell. In certain embodiments, nucleotide coding sequences may be optimized for expression in a select bacteria.
Another aim of the invention may include the expression of a nucleotide coding sequence identified herein generating a protein that may be further isolated and included in a recombinant cell-free expression reaction mixture. In a preferred embodiment, an expressed protein may include, but not be limited to: initiation factor proteins, elongation factor proteins, release factor proteins, ribosome-recycling factor proteins, aminoacyl-tRNA synthetase proteins, and methionyl-tRNA transformylase proteins. Additional nucleotide coding sequences may include RNA polymerase proteins, as well as proteins and compounds identified in the '121 Application related to the inorganic polyphosphate energy-regeneration system incorporated herein.
Another aim of the current invention may include a continuous flow recombinant cell-free expression apparatus. In this preferred embodiment, such a continuous flow recombinant cell-free expression apparatus may include the application of hollow fibers and hollow fiber-based bioreactors as an exchange medium for in vitro transcription, in vitro translation and in vitro biosynthesis of biological, proteins, enzymes, biosimilars and biosynthesis or chemical modification of small molecules using enzymes in a continuous flow operation.
Additional aims of the invention may include one or more of the following preferred embodiments:
Additional aims of the inventive technology may become apparent from the detailed disclosure, figures and claims set forth below.
The accompanying figures, which are incorporated into and form a part of the specification, illustrate one or more embodiments of the present invention and, together with the description, serve to explain certain aspects of the inventive technology. The drawings are only for the purpose of illustrating one or more preferred embodiments of the invention and are not to be construed as limiting the invention.
The present invention is particularly suited for the on-demand manufacturing of therapeutic macromolecules, such as polypeptides, in a cell-free environment that are suitable for direct delivery to a patient. Therefore, the present invention will be primarily described and illustrated in connection with the manufacturing of therapeutic proteins. However, the present invention can also be used to manufacture any type of protein, including toxic proteins, proteins with radiolabeled amino acids, unnatural amino acids, etc. Further, the present invention is particularly suited for the on-demand manufacturing of proteins using cell-free expression, and thus the present invention will be described primarily in the context of cell-free protein expression.
The present invention includes a variety of aspects, which may be combined in different ways. The following descriptions are provided to list elements and describe some of the embodiments of the present invention. These elements are listed with initial embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described systems, techniques, and applications. Further, this description should be understood to support and encompass descriptions and claims of all the various embodiments, systems, techniques, methods, devices, and applications with any number of the disclosed elements, with each element alone, and also with any and all various permutations and combinations of all elements in this or any subsequent application.
The inventive technology described herein may include a novel recombinant cell-free expression system. In one preferred embodiment, the invention may include the generation of a reaction mixture that includes a plurality of core portions that may contribute to the in vitro expression activity. Exemplary core proteins may include the following:
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having one or more initiation factors (IFs). Initiation factors may allow the formation of an initiation complex in the process of peptide synthesis. For example, IF1, IF2 and IF3 may be used in certain embodiments as initiation factors in the reaction mixture. For example, IF3 promotes the dissociation of ribosome into 30S and 50S subunits (i.e., the step being generally needed for initiating translation) and hinders the insertion of tRNAs other than formylmethionyl-tRNA into the P-position in the step of forming the initiation complex. IF2 binds to formylmethionyl-tRNA and transfers the formylmethionyl-tRNA to the P-position of 30S subunit, thereby forming the initiation complex. IF1 may potentiate the functions of IF2 and IF3. In the present invention, it may be preferable to use initiation factors derived from one or more bacteria, and more preferably thermophilic bacteria, for example, those obtained from the bacterial families Bacillaceae, and/or Geobacillus, such as Geobacillus subterraneus, or Geobacillus stearothermophilus. Exemplary amino acid sequences for one or more IFs of the invention may be selected from the group consisting of:
IF1 (SEQ ID NOs. 2, and 70)
IF2 (SEQ ID NOs. 4, and 72)
IF3 (SEQ ID NOs. 6, and 74)
In an embodiment of the invention, one or more of the above amino acid sequence thus comprises at least one IF comprising or consisting of an amino acid sequence encoded by the amino acid sequences according to SEQ ID NOs. 1-2, 4, 6 69-70, 72 and 74, or a fragment or variant of any one of these amino acid sequences. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more IFs according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 1-2, 4, 6 69-70, 72 and 74 disclosed herein.
In the present invention, it may be preferable to use initiation factors expressed in, and/or isolated from one or more bacteria, and more preferably a bacteria configured to express high-levels of proteins, for example, E. coli. Exemplary nucleotide sequences for one or more IFs of the invention may be selected from the group consisting of:
IF1 (SEQ ID NOs. 1, and 69)
IF2 (SEQ ID NOs. 3, and 71)
IF3 (SEQ ID NOs. 5, and 73)
Notably, the nucleotide sequences may be codon-optimized for expression in one or more bacteria, or other protein expression system such as yeast or the like. For example, in this embodiment, exemplary nucleotide sequences SEQ ID NOs. 1, 3 and 5 have been codon-optimized for expression in E. coli.
In an embodiment of the invention, one or more of the above nucleotide sequence thus comprises at least one coding region encoding at least one IF comprising or consisting of a nucleotide sequence encoded by the nucleotide sequence according to SEQ ID NOs. 1, 3, 5, 69, 71, and 73, or a fragment or variant thereof. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more IFs according to the invention may typically comprise a nucleotide sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an nucleotide sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 1, 3, 5, 69, 71, and 73 disclosed herein.
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having one or more elongation factors. An elongation factor, such as EF-Tu, may be classified into 2 types, i.e., GTP and GDP types. EF-Tu of the GTP type binds to aminoacyl-tRNA and transfers it to the A-position of ribosome. When EF-Tu is released from ribosome, GTP is hydrolyzed into GDP. Another elongation factor EF-Ts binds to EF-Tu of the GDP type and promotes the conversion of it into the GTP type. Another elongation factor EF-G promotes translocation following the peptide bond formation in the process of peptide chain elongation. In the present invention, it is preferable to use EFs from bacterial and more preferably from and more preferably thermophilic bacteria, for example, those obtained from the bacterial families Bacillaceae, and/or Geobacillus, such as Geobacillus subterraneus, or Geobacillus stearothermophilus. Exemplary amino acid sequences for one or more EFs of the invention may be selected from the group consisting of:
EF-G (SEQ ID NOs. 8, and 76)
EF-Tu (SEQ ID NOs. 10, and 78)
EF-Ts (SEQ ID NOs. 12, and 80)
EF-4 (SEQ ID NOs. 14, and 82)
EF-P (SEQ ID NOs. 16, and 84)
In an embodiment of the invention, one or more of the above amino acid sequence thus comprises at least one EF comprising or consisting of an amino acid sequence encoded by the amino acid sequences according to SEQ ID NOs. 8, 10, 12, 14, 16, 76, 78, 80, 82, and 84 or a fragment or variant of any one of these amino acid sequences. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more EFs according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 8, 10, 12, 14, 16, 76, 78, 80, 82, and 84 disclosed herein.
In the present invention, it may be preferable to use EFs expressed in, and/or isolated from one or more bacteria, and more preferably a bacteria configured to express high-levels of proteins, for example, E. coli. Exemplary nucleotide sequences for one or more EFs of the invention may be selected from the group consisting of:
EF-G (SEQ ID NOs. 7, and 75)
EF-Tu (SEQ ID NOs. 9, and 77)
EF-Ts (SEQ ID NOs. 11, and 79)
EF-4 (SEQ ID NOs. 13, and 81)
EF-P (SEQ ID NOs. 15, and 83)
Notably, the nucleotide sequences may be codon-optimized for expression in one or more bacteria, or other protein expression system such as yeast or the like. For example, in this embodiment, exemplary nucleotide sequences SEQ ID NOs. 7, 9, 11, 13, and 15 have been codon-optimized for expression in E. coli.
In an embodiment of the invention, one or more of the above nucleotide sequence thus comprises at least one coding region encoding at least one EF comprising or consisting of a nucleotide sequence encoded by the nucleotide sequence according to SEQ ID NOs. 7, 9, 11, 13, 15, 75, 77, 79 and 83 or a fragment or variant thereof. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more EFs according to the invention may typically comprise a nucleotide sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an nucleotide sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 7, 9, 11, 13, 15, 75, 77, 79 and 83 disclosed herein.
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having one or more peptide release factors (RFs). RFs may be responsible for terminating protein synthesis, releasing the translated peptide chain and recycling ribosomes for the initiation of the subsequent mRNA translation. When a protein is synthesized in a release factor-free reaction system, the reaction stops before the termination codon and thus a stable ternary complex (polysome display) composed of ribosome, peptide and mRNA can be easily formed. When a termination codon (UAA, UAG or UGA) is located at the A-position of ribosome, release factors RF1 and RF2 may enter the A-position and promote the dissociation of the peptide chain from peptidyl-tRNA at the P-position. RF1 recognizes UAA and UAG among the termination codons, while RF2 recognizes UAA and UGA. Another termination factor RF3 promotes the dissociation of RF1 and RF2 from ribosome after the dissociation of the peptide chain by RF1 and RF2.
In the present invention, it is preferable to use RFs from bacterial and more preferably from and more preferably thermophilic bacteria, for example, those obtained from the bacterial families Bacillaceae, and/or Geobacillus, such as Geobacillus subterraneus, or Geobacillus stearothermophilus. Exemplary amino acid sequences for one or more RFs of the invention may be selected from the group consisting of:
RF1 (SEQ ID NOs. 18, and 86)
RF2 (SEQ ID NOs. 20, and 88)
RF3 (SEQ ID NOs. 22)
In an embodiment of the invention, one or more of the above amino acid sequence thus comprises at least one RF comprising or consisting of an amino acid sequence encoded by the amino acid sequences according to SEQ ID NOs. 18, 20, 22, 86, and 88 or a fragment or variant of any one of these amino acid sequences. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more RFs according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 18, 20, 22, 86, and 88 disclosed herein.
In the present invention, it may be preferable to use RFs expressed in, and/or isolated from one or more bacteria, and more preferably a bacteria configured to express high-levels of proteins, for example, E. coli. Exemplary nucleotide sequences for one or more RFs of the invention may be selected from the group consisting of:
RF1 (SEQ ID NOs. 17; and 85)
RF2 (SEQ ID NOs. 19; and 87)
RF3 (SEQ ID NO. 21)
Notably, the nucleotide sequences may be codon-optimized for expression in one or more bacteria, or other protein expression system such as yeast or the like. For example, in this embodiment, exemplary nucleotide sequences SEQ ID NOs. 17, 19, and 21 have been codon-optimized for expression in E. coli.
In an embodiment of the invention, one or more of the above nucleotide sequence thus comprises at least one coding region encoding at least one RF comprising or consisting of a nucleotide sequence encoded by the nucleotide sequence according to SEQ ID NOs. 17, 19, 21, 85, and 87 or a fragment or variant thereof. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more RFs according to the invention may typically comprise a nucleotide sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an nucleotide sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 17, 19, 21, 85, and 87 disclosed herein.
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having one or more ribosome recycling factor (RRF) which promotes the dissociation of tRNA remaining at the P-position after the protein synthesis and the recycling of ribosome for the subsequent protein synthesis. In the present invention, it is preferable to use RRFs from bacterial and more preferably from and more preferably thermophilic bacteria, for example, those obtained from the bacterial families Bacillaceae, and/or Geobacillus, such as Geobacillus subterraneus, or Geobacillus stearothermophilus. Exemplary amino acid sequences for one or more RRFs of the invention may be selected from the group consisting of:
RRF (SEQ ID NO. 24, and 90)
In an embodiment of the invention, one or more of the above amino acid sequence thus comprises at least one RRF comprising or consisting of an amino acid sequence encoded by the amino acid sequences according to SEQ ID NOs. 23 and 90 or a fragment or variant of any one of these amino acid sequences. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more RRFs according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 23 and 90 disclosed herein.
In the present invention, it may be preferable to use RRFs expressed in, and/or isolated from one or more bacteria, and more preferably a bacteria configured to express high-levels of proteins, for example, E. coli. Exemplary nucleotide sequences for one or more RRFs of the invention may be selected from the group consisting of:
RRF (SEQ ID NOs. 23, and 89)
Notably, the nucleotide sequences may be codon-optimized for expression in one or more bacteria, or other protein expression system such as yeast or the like. For example, in this embodiment, exemplary nucleotide sequence SEQ ID NO. 23 has been codon-optimized for expression in E. coli.
In an embodiment of the invention, one or more of the above nucleotide sequence thus comprises at least one coding region encoding at least one RF comprising or consisting of a nucleotide sequence encoded by the nucleotide sequence according to SEQ ID NOs. 23, and 89 or a fragment or variant thereof. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more RFs according to the invention may typically comprise a nucleotide sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an nucleotide sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 23, and 89 disclosed herein.
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having one or more aminoacyl-tRNA synthetase (RS) enzymes. Aminoacyl-tRNA synthetase is an enzyme by which an amino acid is covalently bonded to tRNA in the presence of ATP to thereby synthesize aminoacyl-tRNA. In the present invention, it is preferable to use thermophile-origin aminoacyl-tRNA synthetase, for example, those obtained from the bacterial groups Bacillaceae, and/or Geobacillus, or more specifically from the species G. stearothermophilus, or Geobacillus stearothermophilus. Additional embodiments may include the use of an aminoacyl-tRNA synthetase enzymes from a non-thermophile, such as E. coli, such use being in conjunction with aminoacyl-tRNA synthetase enzymes of thermophile origin. Exemplary nucleotide and amino acid sequences for aminoacyl-tRNA synthetase enzymes selected from the group consisting of:
In an embodiment of the invention, one or more of the above amino acid sequence thus comprises at least one RS comprising or consisting of an amino acid sequence encoded by the amino acid sequences according to SEQ ID NOs. 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 134, 126, 128, and 130 or a fragment or variant of any one of these amino acid sequences. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more RSs according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 134, 126, 128, and 130 disclosed herein.
In the present invention, it may be preferable to use RSs expressed in, and/or isolated from one or more bacteria, and more preferably a bacteria configured to express high-levels of proteins, for example, E. coli. Exemplary nucleotide sequences for one or more RSs of the invention may be selected from the group consisting of:
Notably, the nucleotide sequences may be codon-optimized for expression in one or more bacteria, or other protein expression system such as yeast or the like. For example, in this embodiment, exemplary nucleotide sequence SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, and 65 have been codon-optimized for expression in E. coli.
In an embodiment of the invention, one or more of the above nucleotide sequence thus comprises at least one coding region encoding at least one RS comprising or consisting of a nucleotide sequence encoded by the nucleotide sequence according to SEQ ID NOs. 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, and 129 or a fragment or variant thereof. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more RSs according to the invention may typically comprise a nucleotide sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an nucleotide sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, and 129 disclosed herein.
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having a methionyl-tRNA transformylase (MTF). N-Formylmethionine, carrying a formyl group attached to the amino group at the end of methionine, serves as the initiation amino acid in a prokaryotic protein synthesis system. This formyl group is attached to the methionine in methionyl-tRNA by MTF. Namely, MTF transfers the formyl group in Nlυ-formyltetrahydrofolate to the N-terminus of methionyl-tRNA corresponding to the initiation codon, thereby giving a formylmethionyl-tRNA. The formyl group thus attached is recognized by IF2 and acts as an initiation signal for protein synthesis. In the present invention, it is preferable to use an MTF from bacterial and more preferably from and more preferably thermophilic bacteria, for example, those obtained from the bacterial families Bacillaceae, and/or Geobacillus, such as Geobacillus subterraneus, or Geobacillus stearothermophilus. Exemplary amino acid sequences for one or more MTFs of the invention may be selected from the group consisting of:
MTF (SEQ ID NO. 68, and 132)
In an embodiment of the invention, one or more of the above amino acid sequence thus comprises at least one MTF comprising or consisting of an amino acid sequence encoded by the amino acid sequences according to SEQ ID NOs. 68, and 132 or a fragment or variant of any one of these amino acid sequences. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more MTF s according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 68, and 132 disclosed herein.
In the present invention, it may be preferable to use an MTF expressed in, and/or isolated from one or more bacteria, and more preferably a bacteria configured to express high-levels of proteins, for example, E. coli. Exemplary nucleotide sequences for one or more MTFs of the invention may be selected from the group consisting of:
MTF (SEQ ID NO. 67, and 131)
Notably, the nucleotide sequences may be codon-optimized for expression in one or more bacteria, or other protein expression system such as yeast or the like. For example, in this embodiment, exemplary nucleotide sequence SEQ ID NO. 67 has been codon-optimized for expression in E. coli.
In an embodiment of the invention, one or more of the above nucleotide sequence thus comprises at least one coding region encoding at least one MTF comprising or consisting of a nucleotide sequence encoded by the nucleotide sequence according to SEQ ID NOs. 67, and 131 or a fragment or variant thereof. In this context, a fragment of a protein or a variant thereof encoded by the at least one coding region of the one or more MTFs according to the invention may typically comprise a nucleotide sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an nucleotide sequence of the respective naturally occurring full-length protein or a variant thereof, preferably according to SEQ ID NOs. 67, and 131 disclosed herein.
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having a quantity of ribosomes. A ribosome is a particle where peptides are synthesized. It binds to mRNA and coordinates aminoacyl-tRNA to the A-position and formylmethionyl-tRNA or peptidyl-tRNA to the P-position, thereby forming a peptide bond. In the present invention, any ribosome can be used regardless of the origin, however, in a preferred embodiment, ribosomes may be isolated from thermophilic bacteria for use in the recombinant cell-free expression system, and preferably from cell lysates of thermophilic bacteria, such as from the bacterial families Bacillaceae, and/or Geobacillus, such as Geobacillus subterraneus, or Geobacillus stearothermophilus.
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having a quantity of RNA polymerase or fragment or variant thereof which is an enzyme transcribing a DNA sequence into an RNA, occurs in various organisms. As an example, thereof, in one preferred embodiment, the invention may include a T7 RNA polymerase, for example according to amino acid sequence SEQ ID NO. 136. T7 RNA polymerase is derived from the in T7 phage which is an enzyme binding to a specific DNA sequence called T7 promoter and then transcribing the downstream DNA sequence into an RNA. In addition to T7 RNA polymerase, various RNA polymerases are usable in the present invention.
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having a quantity of RNase inhibitor. RNase enzymes promoted the breakdown of RNA into oligonucleotides. RNase inhibitors are known in the art; as such, the type and quantity of RNase inhibitor to be included in a recombinant cell-free expression system is within the skill of those having ordinary skill in the art. Non-limiting examples of RNase inhibitors include mammalian ribonuclease inhibitor proteins [e.g., porcine ribonuclease inhibitor and human ribonuclease inhibitor (e.g., human placenta ribonuclease inhibitor and recombinant human ribonuclease inhibitor)], aurintricarboxylic acid (ATA) and salts thereof [e.g., triammonium aurintricarboxylate (aluminon)], adenosine 5′-pyrophosphate, 2′-cytidine monophosphate free acid (2′-CMP), 5′-diphosphoadenosine 3′-phosphate (ppA-3′-p), 5′-diphosphoadenosine 2′-phosphate (ppA-2′-p), leucine, oligovinysulfonic acid, poly(aspartic acid), tyrosine-glutamic acid polymer, 5′-phospho-2′-deoxyuridine 3′-pyrophosphate P′→5′-ester with adenosine 3′-phosphate (pdUppAp), and analogs, derivatives and salts thereof.
In one embodiment, the recombinant cell-free expression system may include a reaction mixture having a quantity of amino acids, a polynucleotide, such as an mRNA or DNA template encoding a target sequence typically in the form of a plasmid synthesis template, or linear expression (or synthesis) template (LET or LST), and other compounds and sequences identified in the '121 Application related to the inorganic polyphosphate energy-regeneration system, and preferably a coupled AdK/PPK energy regeneration system which may be necessary to energetically drive the in vitro expression reaction.
As generally shown in FIG. 8 of the '121 Application (incorporated herein by reference), in another preferred embodiment, isolated and purified Gst AdK (SEQ ID NO. 8 of the '121 application incorporated herein by reference) and/or TaqPPK (SEQ ID NO. 11 of the '121 application incorporated herein by reference) may be added to this cell-free expression system with a quantity of inorganic polyphosphate. In one embodiment, this quantity of inorganic polyphosphate may include an optimal polyphosphate concentration range. In this preferred embodiment, such optimal polyphosphate concentration range being generally, defined as the concentration of inorganic polyphosphate (PPi) that maintains the equilibrium of the reaction stable. In this preferred embedment, optimal polyphosphate concentration range may be approximately 0.2-2 mg/ml PPi.
As noted above, PPK can synthesize ADP from polyphosphate and AMP. In this preferred embodiment the coupled action of Gst AdK and PPK, may remove adenosine diphosphate (ADP) from the system by converting two ADP to one ATP and one adenosine monophosphate (AMP):
This reaction may be sufficiently fast enough to drive an equilibrium reaction of PPK towards production of ADP:
In this system, the presence of higher concentrations of AMP may further drive the TaqPPK reaction towards ADP.
In a preferred embodiment, the production of macromolecules using the recombinant cell-free system of the invention may be accomplished in a bioreactor system. As used herein, a “bioreactor” may be any form of enclosed apparatus configured to maintain an environment conducive to the production of macromolecules in vitro. A bioreactor may be configured to run on a batch, continuous, or semi-continuous basis, for example by a feeder reaction solution. Referring to FIG. 14 of the '121 application (incorporated herein by reference), in this embodiment the invention may further include a cell-free culture apparatus. This cell culture apparatus may be configured to culture, in certain preferred embodiments thermophilic bacteria. A fermentation vessel may be removable and separately autoclavable in a preferred embodiment. Additionally, this cell-free culture apparatus may be configured to accommodate the growth of aerobic as well as anaerobic with organisms. Moreover, both the cell-free expression bioreactor and cell-free culture apparatus may accommodate a variety of cell cultures, such a microalgae, plant cells and the like.
In one embodiment, the present invention may be particularly suited for operation with a continuous exchange or flow bioreactor (1). In this preferred embodiment, this continuous exchange production apparatus may include a plurality of fibers and hollow fiber-based bioreactor as an exchange medium for in vitro transcription, in vitro translation and in vitro biosynthesis of biologicals, vaccines, proteins, enzymes, biosimilars and biosynthesis or chemical modification of small molecules using enzymes in a continuous flow operation.
Generally referring to
As shown in
In vitro recombinant cell-free expression, as used herein, refers to the cell-free synthesis of polypeptides in a reaction mixture or solution comprising biological extracts and/or defined cell-free reaction components. The reaction mix may comprise a template, or genetic template, for production of the macromolecule, e.g. DNA, mRNA, etc.; monomers for the macromolecule to be synthesized, e.g. amino acids, nucleotides, etc.; and such co-factors, enzymes and other reagents that are necessary for the synthesis, e.g. ribosomes, tRNA, polymerases, transcriptional factors, etc. The recombinant cell-free synthesis reaction, and/or cellular adenosine triphosphate (ATP) energy regeneration system components, incorporated by reference herein, may be performed/added as batch, continuous flow, or semi-continuous flow.
Some of the target proteins that may be expressed by the present invention may include, but not limited to: vaccines, eukaryotic peptides, prokaryotic peptides, bacterial related peptides, fungal related peptides, yeast-related, human related peptides, plant related peptides, toxin peptides, vasoactive intestinal peptides, vasopressin peptides, novel or artificially engineered peptides, virus related peptides, bacteriophage related proteins, hormones, antibodies, cell receptors, cell regulator proteins and fragments of any of the above-listed polypeptides.
Because this invention involves production of genetically altered organisms and involves recombinant DNA techniques, the following definitions are provided to assist in describing this invention.
The terms “isolated”, “purified”, or “biologically pure” as used herein, refer to material that is substantially or essentially free from components that normally accompany the material in its native state or when the material is produced. In an exemplary embodiment, purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A nucleic acid or particular bacteria that are the predominant species present in a preparation is substantially purified. In an exemplary embodiment, the term “purified” denotes that a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. Typically, isolated nucleic acids or proteins have a level of purity expressed as a range. The lower end of the range of purity for the component is about 60%, about 70% or about 80% and the upper end of the range of purity is about 70%, about 80%, about 90% or more than about 90%.
In preferred embodiments, the output of the cell-free expression system may be a product, such as a peptide or fragment thereof that may be isolated or purified. In the embodiment, solation or purification of a of a target protein wherein the target protein is at least partially separated from at least one other component in the reaction mixture, for example, by organic solvent precipitation, such as methanol, ethanol or acetone precipitation, organic or inorganic salt precipitation such as trichloroacetic acid (TCA) or ammonium sulfate precipitation, nonionic polymer precipitation such as polyethylene glycol (PEG) precipitation, pH precipitation, temperature precipitation, immunoprecipitation, chromatographic separation such as adsorption, ion-exchange, affinity and gel exclusion chromatography, chromatofocusing, isoelectric focusing, high performance liquid chromatography (HPLC), gel electrophoresis, dialysis, microfiltration, and the like.
As used herein, the term “activity” refers to a functional activity or activities of a peptide or portion thereof associated with a full-length (complete) protein. Functional activities include, but are not limited to, catalytic or enzymatic activity, antigenicity (ability to bind or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, and the ability to specifically bind to a receptor or ligand for the polypeptide. Preferably, the activity of produced proteins retain at least 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95% or more of the initial activity for at least 3 days at a temperature from about 0° C. to 30° C.
The term “nucleic acid” as used herein refers to a polymer of ribonucleotides or deoxyribonucleotides. Typically, “nucleic acid” polymers occur in either single- or double-stranded form but are also known to form structures comprising three or more strands. The term “nucleic acid” includes naturally occurring nucleic acid polymers as well as nucleic acids comprising known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Exemplary analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). “DNA”, “RNA”, “polynucleotides”, “polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment” are used interchangeably herein. For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). Estimates are typically derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
As used herein, the terms “target protein” refers generally to any peptide or protein having more than about 5 amino acids. The polypeptides may be homologous to, or preferably, may be exogenous, meaning that they are heterologous, i.e., foreign, to the bacteria from which the bacterial cell where they may be produced, such as a human protein or a yeast protein produced in the host bacteria, such as E. coli. Preferably, mammalian polypeptides, viral, bacterial, fungal and artificially engineered polypeptides are used.
As is known in the art, different organisms preferentially utilize different codons for generating polypeptides. Such “codon usage” preferences may be used in the design of nucleic acid molecules encoding the proteins and chimeras of the invention in order to optimize expression in a particular host cell system.
All nucleotide sequences described in the invention may be codon optimized for expression in a particular organism, or for increases in production yield. Codon optimization generally improves the protein expression by increasing the translational efficiency of a gene of interest. The functionality of a gene may also be increased by optimizing codon usage within the custom designed gene. In codon optimization embodiments, a codon of low frequency in a species may be replaced by a codon with high frequency, for example, a codon UUA of low frequency may be replaced by a codon CUG of high frequency for leucine. Codon optimization may increase mRNA stability and therefore modify the rate of protein translation or protein folding. Further, codon optimization may customize transcriptional and translational control, modify ribosome binding sites, or stabilize mRNA degradation sites.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). In addition to the degenerate nature of the nucleotide codons which encode amino acids, alterations in a polynucleotide that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. “Conservative amino acid substitutions” are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine or histidine, can also be expected to produce a functionally equivalent protein or polypeptide. Exemplary conservative amino acid substitutions are known by those of ordinary skill in the art. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
Homology (e.g., percent homology, sequence identity+sequence similarity) can be determined using any homology comparison software computing a pairwise sequence alignment. As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915-9].
According to a specific embodiment, the homolog sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or even identical to the sequences (nucleic acid or amino acid sequences) provided herein. Homolog sequences of SEQ ID Nos 1-22 of between 50%-99% may be included in certain embodiments of the present invention.
The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
A primer is preferably a single-stranded DNA. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 6 to about 225 nucleotides, including intermediate ranges, such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25 to 150 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein.
As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.
The term “reaction mixture,” or “cell-free reaction mixture” or “recombinant cell-free reaction mixture” as used herein, refers to a solution containing reagents necessary to carry out a given reaction. A cell-free expression system “reaction mixture” or “reaction solution” typically contains a crude or partially-purified extract, (such as from a bacteria, plant cell, microalgae, fungi, or mammalian cell) nucleotide translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the translation template. In one aspect, the CF reaction mixture can include an exogenous RNA translation template. In other aspects, the CF reaction mixture can include a DNA expression template encoding an open reading frame operably linked to a promoter element for a DNA-dependent RNA polymerase. In these other aspects, the CF reaction mixture can also include a DNA-dependent RNA polymerase to direct transcription of an RNA translation template encoding the open reading frame. In these other aspects, additional NTPs and divalent cation cofactor can be included in the CF reaction mixture. A reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of ordinary skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of ordinary skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components of the invention. Moreover, those of ordinary skill will understand that some components in a reaction mixture, while utilized in certain embodiments, are not necessary to generate cell-free expression products. The term “cell-free expression products” may be any biological product produced through a cell-free expression system.
The term “about” or “approximately” means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, time frame, temperature, pressure or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” or “approximately” will depend upon the particular system under study. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and includes the endpoint boundaries defining the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
The term “recombinant” or “genetically modified” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, organism, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells may express genes that are not found within the native (nonrecombinant or wild-type) form of the cell or express native genes that are otherwise abnormally expressed, over-expressed, under-expressed or not expressed at all.
As used herein, the term “transformation” or “genetically modified” refers to the transfer of one or more nucleic acid molecule(s) into a cell. A microorganism is “transformed” or “genetically modified” by a nucleic acid molecule transduced into the bacteria or cell or organism when the nucleic acid molecule becomes stably replicated. As used herein, the term “transformation” or “genetically modified” encompasses all techniques by which a nucleic acid molecule can be introduced into a cell or organism, such as a bacteria.
As used herein, the term “promoter” refers to a region of DNA that may be upstream from the start of transcription, and that may be involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter may be operably linked to a coding sequence for expression in a cell, or a promoter may be operably linked to a nucleotide sequence encoding a signal sequence which may be operably linked to a coding sequence for expression in a cell.
The term “operably linked,” when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence. “Regulatory sequences,” or “control elements,” refer to nucleotide sequences that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor or binding sequences; termination sequences; polyadenylation recognition sequences; etc. Particular regulatory sequences may be located upstream and/or downstream of a coding sequence operably linked thereto. Also, particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.
As used herein, the term “genome” refers to chromosomal DNA found within the nucleus of a cell, and also refers to organelle DNA found within subcellular components of the cell. The term “genome” as it applies to bacteria refers to both the chromosome and plasmids within the bacterial cell. In some embodiments of the invention, a DNA molecule may be introduced into a bacterium such that the DNA molecule is integrated into the genome of the bacterium. In these and further embodiments, the DNA molecule may be either chromosomally-integrated or located as or in a stable plasmid.
The term “gene” or “sequence” refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (down-stream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). The term “structural gene” as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
The term “expression,” as used herein, or “expression of a coding sequence” (for example, a gene or a transgene) refers to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., genomic DNA or cDNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).
The term “vector” refers to some means by which DNA, RNA, a protein, or polypeptide can be introduced into a host. The polynucleotides, protein, and polypeptide which are to be introduced into a host can be therapeutic or prophylactic in nature; can encode or be an antigen; can be regulatory in nature, etc. There are various types of vectors including virus, plasmid, bacteriophages, cosmids, and bacteria.
An “expression vector” is nucleic acid capable of replicating in a selected host cell or organism. An expression vector can replicate as an autonomous structure, or alternatively can integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome. Thus, an expression vector are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an “expression cassette.” In contrast, as described in the examples herein, a “cassette” is a polynucleotide containing a section of an expression vector of this invention. The use of the cassettes assists in the assembly of the expression vectors. An expression vector is a replicon, such as plasmid, phage, virus, chimeric virus, or cosmid, and which contains the desired polynucleotide sequence operably linked to the expression control sequence(s).
The terms “expression product” as it relates to a protein expressed in a cell-free expression system as generally described herein, are used interchangeably and refer generally to any peptide or protein having more than about 5 amino acids. The polypeptides may be homologous to, or may be exogenous, meaning that they are heterologous, i.e., foreign, to the organism from which the cell-free extract is derived, such as a human protein, plant protein, viral protein, yeast protein, etc., produced in the cell-free extract. In some embodiment, the term “derived” means extracted from, or expressed and isolated from a bacteria. For example, in one embodiment a protein may be derived from a thermophilic bacteria may mean a protein that is endogenous to a thermophilic bacteria and isolated from said bacteria or expressed heterologously in a different bacteria and isolated as an individual protein or cell extract.
A “cell-free extract” or “lysate” may be derived from a variety of organisms and/or cells, including bacteria, thermophilic bacteria, thermotolerant bacteria, archaea, firmicutes, fungi, algae, microalgae, plant cell cultures, and plant suspension cultures.
As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the culture” includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
The present inventors synthesized and cloned into select expression vectors a plurality of core recombinant proteins, and preferably from a select thermophilic bacteria, for use in a recombinant cell-free expression system. In this embodiment, the present inventors synthesized and cloned into select expression vectors a plurality of core recombinant thermophilic initiation factors (IFs). In this embodiment, the present inventors synthesized and cloned into select expression vectors a plurality of core recombinant thermophilic elongation factors (EFs). In this embodiment, the present inventors synthesized and cloned into select expression vectors a plurality of core recombinant release factors (RFs). In this embodiment, the present inventors synthesized and cloned into select expression vectors at least one core recombinant ribosome recycling factor (RRFs). In this embodiment, the present inventors synthesized and cloned into select expression vectors a plurality of core recombinant aminoacyl-tRNA-synthetases (RSs). In this embodiment, the present inventors synthesized and cloned into select expression vectors at least one core recombinant methionyl-tRNA transformylase (MTF).
As shown generally in Table 1, in one preferred embodiment, the present inventors synthesized, cloned, expressed in E. coli and purified at least twelve (12) different recombinant factors, including nucleotide and/or amino acid sequences, and at least twenty-two (22) recombinant synthetases, including nucleotide and/or amino acid sequences (SEQ ID NOs. 1-132) that form an exemplary Core Recombinant Protein Mixture of at least thirty-four (34) proteins that may be applied to the inventive recombinant cell-free expression system. These core proteins were clone into an expression vector, for example the pET151/D-TOPO (pET151), pET24a(+), or pNAT, as shown in
The present inventors further generated a recombinant cell-free reaction mixture that incorporates one or more of the thirty-four (34) proteins identified, as well as select isolated ribosomes and tRNA from exemplary thermophilic bacteria. The present inventors next included in the recombinant cell-free reaction mixture a quantity of RNA polymerase, and in particular a T7 RNA polymerase enzyme, as well as exemplary amino acids, and buffers. As noted above, the present inventors further generated a recombinant cell-free reaction mixture that incorporates one or more of the components of the inorganic polyphosphate energy-regeneration system identified in the claims of in PCT Application No. PCT/US201 8/012121 ('121 Application).
In one embodiment, the present inventors generated a recombinant cell-free reaction mixture capable of in vitro transcription and translation selected from the group consisting of:
The present inventors confirmed the activity of each purified aminoacyl-tRNA-synthetase (RS). Generally, the aminoacyl-tRNA-synthetase reaction is a two-step process:
Step 1: Activation amino acid+ATP=>aminoacyl-AMP+PPi
Step 2: Transfer aminoacyl-AMP+tRNA=>aminoacyl-tRNA+AMP
The resulting PPi can be measured using the EnzCheck pyrophosphate kit. Utilizing this outline, the present inventors performed kinetic assays using a commercial pyrophosphate assay kit (EnzCheck Pyrophosphate Assay Kit, Molecular Probes, E-6654, incorporated herein by reference). This commercially available assay spectrophotometrically measures indirectly the enzymatic production of pyrophosphate. Each RS reaction was set up in a total of 30 μl with the following final concentrations shown in Table 2. 12.5 μl of the RS reaction mix was used to set up a 50 μl reaction for the pyrophosphate assay as demonstrated in Table 3. Pyrophosphate assays were set up in a 96-well plate and automatically read in 2 min intervals on a plate reader set to read the absorbance at 360 nm. These kinetic measurements were used as a qualitative first test of the activity and functionality of all RS proteins.
Assays were performed according to the manufacturer's instructions and the change in absorbance over time was plotted over time for each RS. As shown in
Resulting AMP from the aminoacyl-tRNA-synthetase reaction can be measured using the AMP-Glo™ kit. The present inventors performed assays using a commercial AMP detection kit (AMP-Glo™ assay, Promega V5012, incorporated herein by reference). This commercially available assay indirectly measures enzymatic production of AMP via a luminescence reaction. An included standard can be used for calibration and calculating the amount of produced AMP. This assay is a quantitative endpoint measurement assay. Each RS reaction was set up in a total of 100 μL with the final concentrations shown in Table 4, and run for one hour at 37° C. Subsequent AMP detection assays were performed in duplicate according to the manufacturer's instructions and produced AMP was calculated using the standard curve (
As an additional confirmation of the activity of each cloned RS, the present inventors performed a malachite green phosphate assay using an available commercial kit (Cayman, Malachite Green Phosphate Assay Kit, #10009325, incorporated herein by reference). Produced pyrophosphate will form a complex with malachite green and lead to a color change which can be measured as absorbance. An included standard can be used for calibration and calculating the amount of produced PPi. This assay is a quantitative endpoint measurement assay. All reactions were performed according to the manufacturer's instructions and the produced PPi was calculated using the standard curve (shown as little inlet on graph).
As shown in Table 4 below, the final concentrations for each RS reaction included a total volume of 150 μl. Exemplary tRNAs from E. coli were utilized in this assay. As shown in
The present inventors demonstrated the production of two exemplary GFP peptides (SEQ ID NO. 134-135) in the invention's recombinant cell-free expression system. As identified in Table 6, a control and template recombinant cell-free expression mixture was generated. Isolation of core recombinant proteins identified in Table 6 below was demonstrated in
The following references are hereby incorporated in their entirety by reference:
[1] Carlson, Erik D. et al. “Cell-Free Protein Synthesis: Applications Come of Age.” Biotechnology advances 30.5 (2012): 1185-1194. PMC. Web. 1 Jan. 2018.
[2] Lloyd, A. J., Thomann, H. U., Ibba, M., & So11, D. (1995). A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity. Nucleic acids research, 23(15), 2886-2892.
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus stearothermophilus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Bacillus sp. X1 (codon-optimized for E. coli)
Bacillus sp. X1
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
E. coli
E. coli
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus (codon-optimized for E. coli)
Geobacillus
Geobacillus stearothermophilus (codon-optimized for E. coli)
Geobacillus stearothermophilus
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus subterraneus DSM 13552 (91A1)
Geobacillus stearothermophilus
Aequorea victoria
Aequorea victoria
This application claims the benefit of and priority to U.S. Provisional Application No. 62/833,555, filed Apr. 12, 2019. The entire specification and figures of the above-referenced application are hereby incorporated, in their entirety by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/28005 | 4/13/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62833555 | Apr 2019 | US |
