SK10862002A3 - Process for producing peptides by using in vitrotranscription/translation system - Google Patents

Abstract

The reaction system from which the peptide is prepared by synthesis in vitro can effectively isolate with high purity and simultaneously is the problem of energy consumption. Method for preparing a peptide or a peptide derivative in the transcription reaction system DNA on RNA and subsequent translation generated RNA, or in the in vitro RNA translation reaction system, wherein part or all of the protein components of the reaction system are labeled with one of a pair of substances adhering to each other and the second substance of the pair is used as an adsorbent to capture labeled protein components after translation.

Description

Technical field

The present invention relates to a method for preparing a peptide or peptide derivative using a DNA to RNA transcription reaction system and subsequent translation of the RNA thus generated or an in vitro RNA translation reaction system (hereinafter referred to as an in vitro transcription / translation reaction system) and a kit of protein components , which contains enzymes and factors for building such a reaction system.

BACKGROUND OF THE INVENTION

Cell-free protein synthesis systems are already known, e.g. systems derived from Escherichia coli, rabbit reticulocytes or wheat germs (Current Opinion in Biotechnology 9: 534-548 (1998), J. Biotechnology 41: 81-90 (1995)).

In these cell-free systems, peptides can be synthesized within a few hours. Proteins can be synthesized in a short time, especially when foreign genes are introduced into and then expressed in host cells (Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), FEBS Letters 414: 268-270 (1997)). In addition, it is recognized or at least expected that protein synthesis in a cell-free system has a number of technical advantages, at least in theory, compared to the case where a foreign gene is introduced into the host cells and then expressed there. The use of these cell-free systems for protein synthesis makes it possible to prepare peptides that would otherwise be cleaved by proteases that originate from host cells, as well as peptides that are toxic to the host cells. These systems can also be used to prepare non-naturally occurring peptide derivatives by incorporating unnatural amino acid residues at specific positions using aminoacyl-tRNAs that carry unnatural amino acid residues (Annu Rev. Biophys. Biomol. Struct. 24: 435). -462 (1995)), or complexes (polysome display) composed of mRNA, ribosomes and peptides. These polysome displays and their uses have been described in He M. et al., J. Immunological Methods 231 (2000) pp. 105-117, Schaffitzel C., J. Immunological Methods 231 (2000) pp. 119-135, Roberts RW., Current Opinion in Chemical Biology 3 (1999) pp. 268-273 and ibid. 9 (1998) s. 534-548 (in particular p. 543 et seq.), And in addition, e.g., FEBS Lett. 450: 105-110 (1999), Proc. Natl. Acad. Sci. USA 95: 14130-14135 (1998) and Proc. Natl. Acad. Sci. USA 94: 4937-4942 (1997).

Although crude cell extracts were used alone in the early stages, only an unstable reaction could be achieved and thus peptides were synthesized only in low yield, namely 0.1 to 0.01% of vital cells. Subsequent studies have elucidated which components contained in the extracts are necessary for gene expression while revealing that unnecessary components and inhibitors (for example endogenous nuclease degrading mRNAs) are present (RNA 6: 1079-1090 (2000)). However, the conventional method of using a cell-free extract as the basis and eliminating unnecessary components from it suffers from the problem that the reaction energy is consumed and thus stops the reaction in about 1 hour of protein synthesis when using a batch system. It is emphasized that factors that cause these problems include the lack of nucleotide triphosphates (Biochim. Biophys. Acta. 1293: 207-212 (1996), J. Biotechnol. 48: 1-8 (1996)), the accumulation of small by-products, such as hydrolyzed triphosphates formed by endogenous enzymes (Biochemistry 22: 346-354 (1983), J. Bi ol. Chem. 260: 15585-15591 (1985)) and energy consumption by factors not necessary for the transcription / translation reaction (J. Ferment. Bioeng. 84: 7-13 (1997), J. Biotechnol. 61: 199-508 (1998)).

The problem of terminating the reaction in a short time can be avoided by continuously supplying the substrate to the peptide / transcription / reaction synthesis system. However, in this case too, another problem arises, namely poor reproducibility. This problem has been solved by clarifying the presence of a germ ribosome inactivator (tritin) and a translation initiation inhibitor in a system using a wheat germ from which these substances have been removed (Bio Industry Vol. 17, No. 5, 20-27 (2000)). However, one more problem remains that such a system consumes considerable energy resources, irrespective of translation.

It has been assumed that these problems encountered in conventional methods are due to the presence of various unknown components in cellular extracts that are not necessary for transcriptional or translational reactions but can hardly be eliminated completely. In this regard, attempts have been made to synthesize peptides in vitro exclusively using enzymes and factors necessary for translation (The Journal of Biological Chemistry Vol. 252, No. 19, 6889-6894 (1997)). For DNA-directed synthesis of β-galactosidase, in addition to E. coli ribosomes, only the following 33 components purified from E. coli extracts were used as factors and enzymes for transcription and translation: RNA polymerase, N-formyltetrahydrofolate, Met-tRNA ^f transformylase, 20 aminoacyl-tRNA synthetases, IF-1, IF-2, IF-3, EF-Tu, EF-G, RF-1 and / or RF-2, CRP, L and L _a. In this study, however, the target product could only be obtained in trace amounts, because at that time insufficient information on the translation mechanism was available and only inadequate purification techniques were available.

Later, Gonza and coworkers constructed an in vitro peptide synthesis system from the pre-charged aminoacyl-tRNA (i.e., having activated activated amino acid) and purified translation factors (Biochem. Biophys. Res. Commun. 126: 792-798 (1985)) . On the other hand, Pavlov and co-workers reconstructed the in vitro translation system using a mixture of partially purified aminoacyl-tRNA synthetase with purified translation factors (Árchives of Biochemistry and Biophysics Vol. 328, No. 1, 9-16 (1996)). They also constructed a fully purified in vitro translation system using short artificial mRNAs (J. Mol. Biol. 273: 389-401 (1997)). However, the successful synthesis of a protein from natural mRNA by means of a translation system containing exclusively the necessary enzymes and factors has never been described so far as it is known by the inventors. However, in conventional cell-free and in vitro peptide synthesis systems that utilize cell extracts, difficult procedures are required to isolate and purify the target peptide product from the protein component of the reaction system, and therefore the target product is obtained with little yield.

SUMMARY OF THE INVENTION

It is an object of the present invention to construct an efficient protein synthesis system in which the energy consumption problem of an in vitro peptide synthesis system can be eliminated, thus providing an in vitro peptide synthesis system wherein the peptide product can be effectively isolated from the reaction system in the peptide. high purity.

The present invention relates to a method of preparing a peptide or peptide derivative using a DNA to RNA transcription reaction system and subsequent translation of generated RNA, or an in vitro RNA translation reaction system, characterized in that part or all of the protein components constituting the transcription / translation reaction system are labeled one of a pair of substances that adhere to each other, and the other is used as an adsorbent to capture the protein component after translation. In this method, a large number of combinations of a substance used to label part or all of the protein components that make up the reaction system with a substance that has been used as an adsorbent to capture the labeled protein components can be used in the transcription / translation reaction system.

Protein components labeled with one of a pair of substances adhering to each other are either part or all of the factors and enzymes for a transcriptional or translational reaction. Specific examples of these factors and enzymes include initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetase, methionyl-tRNA transformylase, and RNA polymerase.

Protein components labeled with one of a pair of substances adhering to each other are the factors and enzymes required for transcription / translation reactions, and other enzymes necessary to form a reaction system. Examples of enzymes necessary to form a reaction system, different from the factors and enzymes required for transcription or translation, are enzymes necessary for energy recovery in the reaction system and enzymes for hydrolysis of the inorganic pyrophosphoric acid (pyrophosphoric) formed during the transcription or translation reaction.

According to the present invention, non-natural peptides carrying non-natural amino acid residues introduced into the desired positions and peptide derivatives such as polysome displays can be prepared using a reaction system for transcribing DNA to RNA and then translation of generated RNA or in vitro translation of RNA that is free of termination factors. .

According to the present invention, the combination of substances that interact in affinity chromatography may be selected from the group consisting of a combination of a protein or peptide fragment with a metal ion, a combination of an antigen with an antibody, a combination of a protein with a protein or a peptide fragment. a molecular weight selected from the group consisting of amino acids, DNA, dyes, vitamins and lectins, combinations of proteins with carbohydrates, and combinations of protein or peptide fragment with an ion exchange resin. In addition, it is preferred to use a combination of a histidine tag (histidine-tag) with a metal chelate such as a nickel complex or a cobalt complex, where the advantage of binding between the protein or peptide fragment and the metal ion is utilized.

Substances that adhere to one another and are useful in the present invention are not limited to combinations of substances that interact with each other in affinity chromatography. For example, substances which adhere to each other under the action of magnetic force may be used.

The present invention further relates to a kit of protein components for a reaction system for preparing a peptide or peptide derivative by transcribing DNA to RNA and then translating the generated RNA or in vitro translation of RNA, which kit is characterized in that it contains part or all of the protein components which form a transcription / translation reaction system, wherein the protein components are selected from the group consisting of enzymes and factors that are labeled with one of a pair of substances adhering to one another. The protein components in this kit are selected from the group consisting of factors and enzymes for the transcriptional or translational reaction, and other enzymes necessary for the formation of the reaction system. Particular examples of factors and enzymes for transcription or translation are initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetase, methionyl-tRNA transformylase, and RNA polymerase. Specific examples of enzymes that are necessary to form a reaction system other than factors and enzymes for transcription or translation are, for example, enzymes for energy recovery in the reaction system and enzymes for hydrolysis of inorganic diphosphoric acid formed during the transcription and translation reaction. The protein component kit of the present invention may comprise an adsorbent for capturing protein components labeled with one of a pair of substances adhering to one another.

The kit of protein components of the present invention may contain mutually different combinations of a substance that is used to label some or all of the protein components of the reaction system with a substance that is used as an adsorbent to capture the labeled protein component.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the ribosomal fraction of E. coli in a sucrose density gradient.

Fig. 2 shows 12% SDS-PAGE profiles of His-tagged initiation, elongation and termination factors stained with Coomassie Brilliant Blue.

Fig. 3A shows the relative DHER activity of His-labeled initiation factors.

Fig. 3B shows optimal concentrations of His-labeled initiation factors expressed as relative DHFR activities.

Fig. 4 shows the activities of His-tagged termination factors expressed as fMFL yields.

Fig. 5A shows a UV absorption diagram at 570 nm of an eluate containing His-labeled SerRS.

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Fig. 5B shows a His-tagged SerRS chromatogram.

Fig. 6 shows 12% SDS-PAGE profiles of His-labeled ARS and MTF.

Fig. 7A shows the optimal concentration of His-tagged termination factors in the in vitro poly (Phe) synthesis system of the present invention expressed as the amount of Phe incorporated.

Fig. 7B shows the progress of the poly (Phe) synthesis reaction (in terms of the amount of Phe incorporated) in the in vitro synthesis system of the present invention and in the S100 extraction system.

Fig. 8A shows 12% SDS-PAGE profiles of DHFR products containing [ ³⁵ S] Met, which were synthesized, respectively, using the in vitro synthesis system of the present invention and the S30 system.

Fig. 8B shows the DHFR activities of these products.

Fig. 9 shows the timing of DHFR synthesis in the in vitro synthesis system of the present invention and in the S30 system.

Fig. 10 shows power consumption in the in vitro synthesis system of the present invention (right) and the S30 (left) system over time.

Fig. 11 shows the generation of DHFR that carries a valine residue incorporated at position 37 as a model for incorporating a non-natural amino acid in the in vitro synthesis system of the present invention.

Fig. 12A shows a UV absorption diagram at 570 nm of an eluate containing His-tagged T7RNA polymerase.

Fig. 12B shows a chromatogram of His-labeled T7RNA polymerase.

Fig. 13 shows the SDS-PAGE profiles of various proteins synthesized using the in vitro synthesis system of the present invention.

Fig. 14 shows the purity of DHFR, which is a translation product of the in vitro synthesis system of the present invention, after passing through an ultrafiltration membrane with a transition limit of 100 kDa and a nickel column. The arrow in this figure shows the DHFR position.

DETAILED DESCRIPTION OF THE INVENTION

The in vitro synthesis system of the present invention is a reaction system for synthesizing peptides by transcribing DNA to RNA and subsequently translation of generated RNA or translation of RNA without the use of cells alone.

The term peptide as used herein means a substance composed of 2 or more natural or unnatural amino acids linked together via a peptide bond and includes both oligopeptides and polypeptides. Moreover, proteins having a specific three-dimensional structure of polypeptides are also within the scope of this term.

The term RNA, as used herein, also includes the categories of artificial RNA and mRNA, as well as the term DNA, as used herein, also includes the categories of artificial DNA and cDNA.

The in vitro synthesis system of the present invention, wherein the DNA is transcribed to RNA and then the generated RNA is translated or RNA is translated, is a reaction system existing in prokaryotic cells or eukaryotic cells, consisting of ribosomes, factors and enzymes for transcriptional or translational reactions , and other enzymes required to assemble the reaction system, various substrates, buffers, and salts. Although the excellent effects of the present invention can be established in a reaction system that has been artificially reconstituted, the present invention is also applicable to reaction systems where some of these components are added in the form of a cell extract.

Factors and enzymes for transcriptional or translational reactions are not limited to those derived from prokaryotic cells such as E. coli, but can also be used that originate from eukaryotic cells. In the case of (1) RNA translation, these factors and enzymes include initiation factors, elongation factors, termination factors, 20 aminoacyl-tRNA synthetases and tRNAs bound with a natural or unnatural amino acid, and if the in vitro E. coli-derived reaction system still includes methionyl- tRNA transformylase. In the case of (2) transcription

DNA to RNA and then translation of generated RNA, these factors and enzymes include, in addition to those mentioned in (1), an RNA polymerase, such as T7RNA polymerase. The translational reaction can be regulated by eliminating termination factors from the reaction system described in case (1) or case (2), as discussed further below.

Examples of enzymes other than factors and enzymes for transcriptional or translational reactions include enzymes for energy recovery in the reaction system such as creatine kinase, myokinase and nucleoside diphosphate kinase (NDK) and enzymes that hydrolyze inorganic diphosphoric acid (pyrophosphoric or pyrophosphoric) translation reactions such as inorganic pyrophosphatase.

Examples of various substrates are natural amino acids, non-natural amino acids, nucleotide triphosphates as energy sources, creatine phosphate and formylfolic acid. Nucleotide triphosphates include ATP, GTP, CTP, and UTP. ATP and GTP are used in the above case (1), while ATP, GTP, CTP and UTP are used in the above case (2).

As a buffer, a potassium phosphate solution (pH 7.3) is usually used. As the salt, for example, potassium glutamate, ammonium chloride, magnesium acetate, calcium chloride, putrescine, spermidine and dithiothreitol (DTT) are commonly used. It will be appreciated that other suitable components may be included in the reaction system in addition to the above components.

A first feature of the present invention is that in a peptide synthesis system by transcribing DNA to RNA and then translating the resulting RNA or in vitro translation of RNA, some or all of the protein components of the transcription / translation reaction system are labeled with one of two adhering substances and the substance is used as an adsorbent to capture the labeled protein components after translation. Thus, the target peptide can be readily separated from the protein component of the reaction system and obtained in an extremely high purity.

Histidine tagging is sometimes used to prepare and purify the individual protein components of the reaction system, namely, factors and enzymes for a transcription / translation reaction. For example, the use of a histidine tag in the preparation and purification of EF-Tu elongation factors is described (Eur. J. Biochem, 210: 177-183 (1992)), EE-G (Cell 92: 131-139 (1998)) and EE- TS (Archives of Biochemistry and Biophysics 348: 157-162 (1997)), the preparation and purification of the RF2 termination factor (Proc. Natl. Acad. Sci. USA 95: 8165-8169 (1998)) and the preparation and purification of phenylalanyl-tRNA synthetase (Protein Expression and Purification 8: 347-357 (1996)). In these cases, however, production and purification were not performed in order to prepare an in vitro peptide synthesis system, but only to investigate the function or properties of individual proteins.

To separate a protein or protein derivative produced by expressing a gene in a transformant, such as E. coli, is often used e.g. affinity chromatography using a combination of a histidine tag and a nickel column, a combination of glutathione-S-transferase and a giutathion-sepharose resin column, or a combination of an epitope label and an antibody. In such cases, a residue capable of selectively binding to an adsorption column is usually incorporated into the target peptide. In cell-free systems using commercially available cell extracts, a vector is used to incorporate a histidine tag into a target peptide. In this case, the product is obtained in the form of a fusion protein of the target peptide with a histidine tag, which should be enzymatically removed from the peptide after synthesis.

Unlike conventional methods, the present inventors have introduced one of a pair of substances adhering to one another, not into the target peptide, but into the protein components that make up the in vitro peptide synthesis system based on the new discovery that a transcriptional or translational reaction may occur. even if the factors and enzymes for transcription or translation and other enzymes are labeled with one of a pair of substances that adhere to each other.

The combination of a pair of substances adhering to each other for use in the present invention can be selected in virtually any choice as long as the transcription or translation reaction is not impaired. Although the adhesion of these two substances to one another may be reversible or irreversible, it is preferable to use a pair of substances which adhere to one another reversibly. This is because the protein components that make up the reaction system can be reused in such a case.

An example of a combination of adhering substances may be a combination of an adsorption column with a substance capable of selectively binding to the adsorption column. Typical examples include substances that interact with each other in affinity chromatography. For example, a metal complex, such as a nickel or cobalt complex, serves as a ligand for an adsorption column while a histidine tag (His-tag) serves as a substance capable of selectively binding to an adsorption column. In addition, combinations of different ligands with agents capable of selectively binding to these ligands may be used, as further detailed below, as long as the reaction is not disrupted. Thus, combinations of agents interacting in affinity chromatography suitable for use in the present invention may be selected from the group consisting of, for example, a combination of a protein or peptide fragment with a metal ion, a combination of an antigen with an antibody, a combination of a protein with a protein or a peptide fragment. a specific low molecular weight compound selected from the group consisting of amino acids, DNA, dyes, vitamins and lectins, a combination of a protein with a carbohydrate, and a combination of a protein or peptide fragment with an ion exchange resin.

The cross-adhering substances suitable for use in the present invention are not limited to combinations of a substance interacting in affinity chromatography, but can be arbitrarily selected depending on the purpose chosen. For example, substances may be used; which adhere magnetically to each other. An example is a combination of a protein labeled with magnetic beads and a magnet. In this case, the protein components of the peptide synthesis system, which have been individually labeled with beads, can be adsorbed by a magnet and thus captured.

In the present invention, the adsorbent is used in the form of, for example, a column, matrix, filter, or beads. Alternatively, it can be fixed to a carrier if desired. For fixing the adsorbent to the carrier, a suitable composition may be selected from known techniques depending on the properties of the adsorbent.

There are a large number of combinations of a substance to label part or all of the protein components of the reaction system with the substance that has been used as an adsorbent to capture the labeled protein component. Various combinations may be used in the individual reaction systems. A preferred procedure may be considered first to select the most suitable labels for the corresponding protein components and then to select adsorbents suitable for these labels.

Since, according to the present invention, the factors and enzymes for the transcription / translation reaction system and other enzymes are labeled with one substance from a pair of substances adhering to each other, the protein components constituting the reaction system can be obtained individually, each in high purity. and an unnecessary or inhibitory component. Thus, the reaction system can be assembled and the efficiency of the reaction can be greatly increased. In addition, the target peptide can be rapidly separated from the reactants after peptide synthesis. In conventional cell-free systems, the peptides formed are purified by extraction. Thus, it is necessary to select a suitable purification procedure for each case, depending on the physical and chemical properties of the reaction product. In contrast, in the present invention, the constituents of the reaction system are eliminated by the use of an adsorbent and thus the reaction product is purified. It is also at least theoretically possible to use the same purification procedure for any reaction product, regardless of its physical and chemical properties. In addition, a high purity target peptide is obtained.

Further, according to the present invention, the components of the reaction system can be reliably controlled so that the reaction system can be built without termination factors. Due to this feature, the present invention allows the construction of polysome displays of various types, thereby expanding the application possibilities of the in vitro peptide synthesis system. Specifically, ternary complexes (polysome displays) assembled from a peptide, RNA and ribosome, can be obtained by expressing various DNA or RNA in an in vitro synthesis system of peptides without termination factors according to the present invention. By separating these polysome displays from other complexes using the peptide as the target, the target peptide and RNA can be obtained simultaneously. By applying product termination factors, the corresponding RNA can be obtained. In this case, the peptide that has been cut from the ribosome and the corresponding RNA can be readily isolated by the action of termination factors labeled with one of a pair of mutually adhering substances. For example, the corresponding DNA can be obtained from an isolated polysome display by RT-PCR as described in Current Opinion in Biotechnology 9: 534-548 (1998). By decomposing this polyzomal display with EDTA, RNA can be recovered. It is particularly technically preferred that RNA or DNA corresponding to the selected target peptide can be readily obtained by random expression. Although the seminal expression of DNA and RNA in a cell-free system using a kit containing rabbit reticulocytes is already known, these are very complicated procedures (WO91 / 05058).

Since the components of the reaction system can be reliably controlled, it is possible to synthesize a peptide that contains unnatural amino acid residues using an in vitro peptide synthesis system. In particular, a peptide having non-natural amino acid residues can be synthesized by the method of the present invention as follows. A suppressor tRNA with an unnatural amino acid residue attached and corresponding to a termination codon different from the C-terminal termination codon is added to the reaction system. Then, the DNA or RNA that has been modified by insertion of the termination codon of the corresponding suppressor tRNA at the position to incorporate the non-natural amino acid residue is transcribed or translated, thereby providing a peptide having the non-natural amino acid residue incorporated. More particularly, such synthesis can be carried out as follows. To incorporate a non-natural amino acid residue, one of the termination codons (UAA, UAG and. UGA), for example UGA or UAG, is inserted at a desired position within an open reading frame (ORF) and the UAA codon is used to terminate translation. Then, the suppressor tRNA carries the anticodon to UGA and / or UAG. it is prepared by in vitro transcription and is bound to it by a non-natural amino acid. Thereafter, the method of the present invention is performed using this RNA and suppressor tRNA as described for translation of RNA. Thus, a peptide having a non-natural amino acid residue inserted at a specific point can be synthesized. Alternatively, the peptide may be synthesized by separate synthesis of the corresponding DNA followed by transcription and translation.

The use of a reaction system obtained by eliminating termination factors from the described reaction system makes it possible to obtain a polysome display consisting of a peptide, mRNA and a ribosome that carries one or more unnatural amino acid residues at the desired positions. By the action of termination factors labeled with one of a pair of substances adhering to each other on this polysome display, the peptide that has been separated from the ribosome and the corresponding RNA can be readily isolated. For example, the corresponding DNA can be obtained from the polysome display by RT-PCR as described in Current Opinion in Biotechnology 9: 534-548 (1998). RNA can also be obtained by dissociating the polyzomal display by EDTA.

Thus, peptide derivatives prepared by the method of the present invention include polyzomal displays and unnatural peptides having unnatural amino acid residues at the desired positions.

The process for preparing the peptide or peptide derivative of the present invention can be accomplished using a delivery system in a conventional manner. Alternatively, it can be carried out using a variety of known methods, such as e.g. flow method, wherein materials including substrates are continuously fed to the reaction or the reaction product is occasionally removed or by dialysis (see, e.g., Japanese Patent 110236/1995, Tanpakushitsu, Kakusan, Koso (Proteins, Nucleic Acids and Enzymes) Vol. 44, No 4, 598-605 (1999), Current Opinion in Biotechnology 9: 534-548 (1998)).

Hereinafter, the present invention will be illustrated in more detail with reference to the following examples. However, it is to be understood that the invention is not limited to these examples.

(1) Peptides and peptide derivatives that can be prepared according to the invention

Natural peptides and non-natural peptides of any type can be prepared by the method of the present invention. It can be said that the use of the method of the invention makes it possible to prepare peptides that would be cleaved by host cell proteases such as dihydrofolate reductase (DHFR), lysozyme (phage λ derived), green fluorescent protein (GFP), as well as peptides that are host cells toxic.

The term natural peptides, as used herein, means peptides composed of 20 natural (essential) amino acids that are encoded by the genetic code, while peptides containing other amino acids are called non-natural peptides.

In addition, ternary complexes (polysome displays) composed of peptide, RNA and ribosome can be readily obtained using a reaction system without a termination factor in the preparation method of the present invention.

Examples of non-natural peptides that can be prepared by the method of the present invention include peptides having modified natural amino acids, modified uncharged amino acids, modified acidic amino acids, modified basic amino acids, non-α-amino acids, φ, φ-angle replacement amino acids and amino acids having functional groups selected from the group consisting of nitro, amidine, hydroxylamine, quinone, aliphatic compounds, and cyclic and unsaturated hydrocarbon groups. Methods of synthesizing such peptides in cell-free protein synthesis systems using cell extracts are known (see, for example, JP-W-Hei-4-504651 / WO90 / 05785). As a specific example of these unnatural peptides, DHFR having a protected cysteine residue inserted at a suitable position, such peptide does not occur in nature. Further, mention may be made of peptides having unnatural amino acid residues, such as p-fluorophenylalanine, p-nitrophenylalanine or homophenylalanine, inserted at the desired position. It is also known that the β-lactamase variant having these 3 non-natural amino acid residues inserted as phenylalanine substituents at position 66 exhibits sufficient enzymatic activity (Bio Industry 8: 749-759 (991)). Such non-natural peptides may also be readily prepared by the method of the present invention.

(2) Ribosomes

A ribosome is a particle where peptides are synthesized. It binds to mRNA and coordinates aminoacyl-tRNA to the A-site and formylmethionyl-tRNA or peptidyl-tRNA to the β-site to form a peptide bond (Sience 289: 920-930 (2000)). Any ribosome, regardless of its origin, may be used in the practice of the present invention as long as it retains the function described. Although E. coli ribosomes are commonly used, eukaryotic ribosomes can also be used. Preferably, ribosomes from E. coli are used in the present invention, for example ribosomes obtained from an E. coli A19 strain or an MRE 600 strain.

(3) Factors and enzymes for transcription or translation for use in the in vitro peptide synthesis system of the present invention (3-1) Initiation Factors

Initiation factors are factors that are essential to or significantly support the formation of a complex in the peptide synthesis process. IF1, IF2 and IF3 are known as initiation factors derived from E. coli (Biochemistry 29: 5881-5889 (1990)). IF3 promotes the dissociation of the ribosome into the 30S and 50S subunits (i.e., a step necessary for translation initiation) and prevents insertion of a non-formylmethionyl-tRNA tRNA into the P-site to form an initiation complex. IF2 binds to formylmethionyl-tRNA and thus transfers formylmethionyl-tRNA to the β-site of the 30S subunit, thereby forming an initiation complex. IF1 enhances the functions of IF2 and IF3. Initial factors derived from E. coli, for example those derived from an E. coli K12 strain, may advantageously be used in the present invention. However, eukaryotic initiation factors may also be used.

(3-2) Elongation factors

The elongation factor EF-Tu is classified into two types, i. GTP and GDP. GTP-type EF-Tu binds to aminoacyl-tRNA and is transferred to the ribosome A-site. When EF-Tu is released from the ribosome, GTP is hydrolyzed to GDP (EMBO J. 17: 7490-7497 (1998)). Another elongation factor of EF-Ts binds to the EF-Tu GDP type and stimulates its conversion to the GTP type (Archives of Biochemistry and Biophysics 348: 157-162 (1997)). Another elongation factor EF-G promotes translocation following peptide bonding in the peptide chain elongation process (Nature Structural Biology 6: 643-647 (1999), FEMS Microbiology Reviews 23: 317-333 (1999)). According to the present invention, it is preferred to use E. coli-derived elongation factors, for example elongation factors prepared from an E. coli K12 strain. However, it is also possible to use eukaryotic elongation factors.

(3-3) Termination factors

Termination factors are essential in the process of terminating protein synthesis, releasing the translated peptide chain, and recycling ribosomes to initiate further translation of mRNA. When a protein is synthesized in a reaction system without a termination factor, the reaction stops before the termination codon and thus easily forms a stable ternary complex (polysome display) consisting of ribosome, peptide and mRNA. The non-natural amino acid is introduced into the peptide chain by eliminating either RF1 or RF2 from the reaction system. Thus, the non-natural amino acid is intraporated with high efficiency at the UAG codon in the case of RF1 elimination or at the UGA codon in the case of RF2 elimination. When the termination codon (UAA, UAG or UGA) is located at the A-site of the ribosome, the termination factors RF1 and RF2 enter the A-site and stimulate dissociation of the peptide chain from the peptidyl-tRNA at the P-site. RF1 recognizes UAA and UAG from the termination codons, while RF2 recognizes UAA and UGA. Another termination factor RF3 promotes the dissociation of RF1 and RF2 from the ribosome after dissociation of the peptide chain by RF1 and RF2. The ribosome recycling factor (RRF) aids in the dissociation of the tRNA remaining at the P-site after protein synthesis and recycling of the ribosome for further protein synthesis. According to the present invention, one of the termination factors is RRF. The functions of termination factors RF1, RF2, RF3 and RRF have been described in EMBO J. 16: 4126-4133 (1997) and EMBO J. 16: 4143-4141 (1997). Terminating factors derived from E. coli can be advantageously used according to the present invention, for example termination factors prepared from an E. coli K12 strain. However, it is also possible to use eukaryotic termination factors.

(3-4) Aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetase is an enzyme by which an amino acid is covalently linked to tRNA in the presence of ATP, thereby synthesizing aminoacyl-tRNA (RNA 3: 954-960 (1997), Tanpakushitsu, Kakusan, Koso (Proteins, Nucleic Acids and Enzymes) 39 1215-1225 (1994)). According to the present invention, an aminoacyl-tRNA synthetase derived from E. coli, for example from an E. coli K12 strain, may be advantageously used. However, eukaryotic aminoacyl-tRNA synthetase can also be used.

(3-5) Methionyl-tRNA transformylase

N-Formylmethionine (fMet), which carries a formyl group attached to the amino group at the end of methionine, serves as an initiating amino acid in the prokaryotic protein synthesis system. This formyl group is attached to methionine in methionyl-tRNA by methionyl-tRNA transformylase (MTF). In particular, methionyl-tRNA transformylase transfers the formyl group from N ¹⁰ -formyltetrahydrofolate to the N-terminus of the methionyl-tRNA corresponding to the initiation codon, thereby forming formylmethionyl-tRNA (Proc. Natl. Acad. Sci. USA 96: 875-880 (1999)). The formyl group so bonded is known by IF2 and thus acts as an initiation signal for protein synthesis. Although MTF does not occur in the synthetic system in the cytoplasm of eukaryotes, it is present in the eukaryotes in the synthetic system of mitochondria and chloroplasts. According to the present invention, β-derived methionyl-tRNA transformylase can be advantageously used. coli, for example obtained from an E. coli K12 strain.

(3-6) RNA polymerase

It is also known that RNA polymerase, an enzyme that transcribes DNA sequences to RNA sequences, occurs in various organisms. By way of example, T7RNA polymerase derived from T7 phage, an enzyme that binds to a specific DNA sequence called the T7 promoter and then transcribes a DNA sequence that lies downstream of the RNA. The present inventors attached a histidine tag to the N-terminus of the T7RNA polymerase and thus expressed the fusion protein in large quantities in E. coli strain BL21. Then the expression product was purified by affinity chromatography using a nickel column. The His-tagged T7RNA polymerase thus prepared is novel. In addition to T7RNA polymerase, various other RNA polymerases are suitable for use in the present invention. For example, commercially available T3 RNA polymerase and SP6 RNA polymerase can be used.

(3-7) Aminoacyl-tRNA linked to a non-natural amino acid

By incorporating amino acid residues other than the 20 essential amino acids that make up natural proteins into proteins, the intrinsic functions of the proteins can be improved or new useful functions or characteristics can be assigned to them. Aminoacyl-tRNA with bound non-natural amino acid can be prepared by synthesis of a 3-terminal CA-deficient suppressor tRNA via in vitro transcription and its ligation using RNA ligase to a chemically synthesized aminoacyl-pCpA having a non-natural amino acid (Baiosaiens

Indasutori (Bioscience and Industry) 47: 16-24 (1989)).

(4) Enzymes necessary for the assembly of the reaction system, in addition to factors and enzymes for transcription or translation, for use in the in vitro peptide synthesis system of the present invention (4-1) Enzymes for energy recovery in the reaction system

Examples of this type include creatine kinase, myokinase, and nucleoside diphosphate kinase (NDK). Creatine kinase, called creatine phosphokinase (CPK), catalyzes the transfer of a phosphate group from ATP to creatine.

Myokinase, also called adenylate kinase, is involved in the formation of ATP from ADP and in the simultaneous generation of AMP. NDK catalyzes the transfer of the γ-phosphate group between nucleoside diphosphate and nucleoside triphosphate. According to the present invention, these enzymes derived from E. coli can be advantageously used, for example from the E. coli K12 strain. However, eukaryotic enzymes may also be used.

(4-2) Enzymes for the hydrolysis of inorganic pyrophosphoric acid (pyrophosphoric acid) produced by a transcriptional or translational reaction

An example of an enzyme of this type is inorganic pyrophosphatase. According to the present invention, an enzyme derived from E. coli, for example from an E. coli K12 strain, may be advantageously used. However, a eukaryotic enzyme may also be used.

Of the components of the reaction system as described in (3) and (4), the protein components are expressed in large quantities in E. coli (e.g., in a commercially available E. coli BL21 strain) in the form of fusion proteins (e.g., Hisoidin-labeled proteins) labeled at the N- or C-terminus with one of a pair of substances adhering to each other, as described in more detail below. The proteins thus expressed are then purified using a nickel column coupled to an adsorbent containing a second substance, such as by FPLC, and then fed to the reaction system. In addition to E. coli, these proteins can also be expressed in animal cells, yeast, Bacillus subtilis, and other expression systems. Alternatively, these proteins can be prepared using an in vitro peptide synthesis system.

(5) Labeling compound (label) and adsorbent, i. j. a pair of adhering substances

In the present invention, all or part of the protein components that make up the reaction system, as described in (3) and (4), are labeled by one of a pair of substances adhering to each other and these labeled protein components are captured by the other substance as adsorbent. a target peptide readily isolated from the reaction system. As a typical example of a pair of mutually adhering substances, there may be mentioned substances interacting with each other in affinity chromatography. However, any pairs of adhering materials may be used in the present invention, i. not only substances that interact with each other in affinity chromatography when these substances can be used to capture the protein component.

Proteins exert their physiological effects through specific interactions with certain substances · Adsorption chromatography, which is performed using this specific interaction (affinity) between the protein and a particular substance (ligand), is called affinity chromatography. An example of a combination of a substance that adheres specifically to a second substance is e.g. a protein or peptide fragment with a metal ion or chelate compound, an antigen with an antibody, a cytokine or hormone with an appropriate receptor, and an enzyme with a substrate or inhibitor. In addition, specific amino acids, DNA, dyes, vitamins, lectins and the like. they bind to proteins having affinity for them.

One of the materials of such a combination is fixed as a ligand on a carrier or support to form an adsorbent. Then, the materials labeled with the second substance (i.e., in the case of the present invention, the protein components that make up the reaction system) pass (filtered) through the carrier. Thus, the label specifically binds to a ligand. Affinity chromatography based on such specific binding has already been widely used as a method of protein purification. Many manufacturers sell different types of carriers, so they are available in a very wide range. For purification of proteins based, for example, on an antigen-antibody reaction, a combination of an antigenic determinant (epitope) having a known structure with an antibody that is specific for that epitope is used. Various combinations of vectors and adsorbents are available on the market for carrying out this method. In a preferred embodiment of the present invention, a combination of substances that adsorb to each other and used in affinity chromatography is used. In preparing labeled protein components for use in the present invention, the label is useful for purification. It is also possible to label the protein components with a plurality of labels simultaneously. In such a case, it is possible that the label usable for purification is removed in the preparation process, while other labels are used to separate the target product prepared in the in vitro peptide synthesis system.

Embodiments of the present invention will be further illustrated by providing some examples of combinations of adhering materials. However, it is to be understood that the present invention is not limited to these examples.

(5-1) A method using the binding of a protein or peptide fragment to a metal ion or chelate compound.

A. His-tag with a metal complex, such as a nickel or cobalt complex

In practice, the binding of His-tag to a metal complex, such as a nickel or cobalt complex, is commonly used to purify proteins. Specifically, the His-tag is ligated to the DNA from which the fusion protein having the His-tag is to be expressed. Then, the fusion protein is captured and purified using a column containing, for example, a nickel, cobalt, copper or zinc complex. The protein can then be eluted from the column using an eluent containing imidazole (see, for example, Tanpakushitsu Jikken Noto (Protein Experiment Note) (I), p.139, Ch. 5: 1). His-Tag Tanpakushit no Hatugen to Seisei of His-Tag Protein), Yasumitsu and Wakui, Yodosha, JA Bornhorst and JJ Falke, Purification of Proteins Osing Polyhistidine Affinity Tags, Methods in Enzymology 326: 245-254, (2000), Proteins 41: 144-53 (2000) , FEMS Microbiol. Lett. 188: 147-51 (2000), and J. Bacteriol. 182: 4304-9 (2000)). These combinations are suitable for use in the present invention. For the expression of genes that encode His-tagged protein components, as known, e.g. E. coli cells (MWVan. Dyke M. Sirito and M. Sawadogo, Gene 111: 95, 1992), yeast Saccharomyces cerevisiae (DC Kaslow and J. Shiloach, Bio / Technology 12: 494, 1994), mammalian cells ( R. Jankencht and A. Nordheim, Gene 121: 321, 1992) as well as baculovirus-infected insect cells (A. Kuusin, M. Arvola, C. Oker-Blom, and K. Keinanen, Eur. J. Biochem. 233: 720, 1995). All of these cells can optionally be used in the present invention.

The following protocol roughly shows an example of a method for purifying a histidine-labeled protein component using a His-tag and a nickel column. A number of variations of this method are known, and thus a suitable variation can be readily selected from them.

1. A His-tag consisting of 6 His residues is ligated at the N-terminus of the target protein by genetic engineering (recombinant DNA) methods to obtain a fusion protein.

2. By sonication on ice, cells that express the labeled protein are broken and the broken cells are resuspended in loading buffer (300 mM NaCl, 50 mM NaH ₂ PO ₄ , pH 8.0).

3. Centrifuge the cell lysate (30,000 g, 4 ° C, 30 minutes).

4. Add 50% Ni ²⁺ -NTA (Qiagen) slurry pre-equilibrated in ice-cold loading buffer to the supernatant. Stir at 4 ° C for 1 hour.

5. Load the resin onto the column. The column is washed with 20 volumes of loading buffer at 4 ° C.

6. Wash the column with 20 volumes of loading buffer (containing 10 mM imidazole, pH 8.0) at 4 ° C.

7. The target protein is eluted with 20 volumes of loading buffer with a concentration gradient of 10 to 250 mM imidazole in loading buffer. A 1 ml fraction is collected and the target protein is identified by SDS-PAGE.

B. Thioredoxin with phenylarzine oxide (PAO)

In this method, a fusion protein composed of a target protein and thioredoxin is prepared and adsorbed onto a PAO-fixed agarose gel (TioBond ™ resin, Invitrogen), preferably utilizing the binding of thioredoxin to PAO, followed by elution with β-mercaptoethanol (β-ΜΕ) (A-ΜΕ). Alejo, RJ Yanez, JM Rodriguez, E. Vinuela, and ML Salas, African Swine Fever Trans-Prenyltransferase Virus, The Journal of Biological Chemistry 272: 9417-9423, 1997). This method is also suitable for use in the present invention.

The following protocol roughly shows an example of protein purification using the above method.

1. A culture of transformed cells (E. coli, pTrxus vector, Invitrogen) grown overnight was diluted 20-fold in RM medium (0.6 NaH 2 PO ₄ , 0.3% KH 2 PO 4, 0.05% NaCl, 0.1% NH 4 Cl, 2% casamino acid, 0.0095% MgCl 2) containing 100 µg / ml (final concentration) ampicillin and incubated at 30 ° C.

2. After incubation and reaching Α _{50 50} = 0.5, 100 µg / ml (final concentration) of tryptophan was added to induce expression of the fusion protein. The incubation was then continued at 34 ° C for a further 2 hours.

3. Cells were harvested by centrifugation. The cell pellet was resuspended in 5 ml assay buffer (100 mM Tris-HCl (pH 7), 150 mM NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol). Then the cells were broken by sonication.

4. The cell suspension was centrifuged (10,000 g, 15 min) and then the supernatant was collected.

5. The supernatant was incubated with 2 ml of ThioBond ™ resin at 4 ° C for 60 minutes to bind the fusion protein contained in the supernatant to the resin.

6. The column was packed and washed with 30 volumes of assay buffer (100 mM Tris-HCl (pH 7), 150 mM NaCl, 1 mM EDTA, 20 mM β-mercaptoethanol). and

7. The target protein was eluted with assay buffer in a β-mercaptoethanol gradient.

(5-2) A method using the binding of an antigen or antigenic fragment (epitope-tag) to an antibody

A. T7-tag and monoclonal antibody specific for T7-tag

The T7-tag is a sequence of 11 amino acids from gene 10 that originates from T7 phage. The combination of the T7-tag with an antibody against this sequence is used as a means for protein purification. Specifically, this purification process involves binding a DNA sequence that encodes a T7-tag to a gene, then expressing the target protein and capturing and purifying the T7-tag fusion protein using a monoclonal antibody that is specific for the T7-tag as an adsorbent. An example of an adsorbent is commercially available agarose with a T7-tag antibody (T7-Tag Antibody Agarose, from Novagen). Citric acid is used as eluent. This combination is also suitable for use in the present invention (see R. Deora, T. Tseng, and TK Misra, Alternative Transcription Factor, and ^SB of Staphylococcus aureus: Characterization and Role in Transcription of the Global Locus Controllers sar. Journal of Bacteriology 179: 6355-6359, 1997).

The following protocol briefly shows an example of a process for purifying the T7-labeled protein component. There are a number of variations of this process, some of which are easy to select.

1. Transformed E. coli cells / pET vector (Novagen) in 2xYT medium (1.6% Bacto Trypton, 1% yeast extract, 0.5% NaCl, 0.4% glucose) containing 20 gg / ml chloramphenicol and 30 μς / πιΐ kanamycin.

2. After incubation and reaching A ₆ O = 0/6, IPTG is added to induce target protein expression. The incubation is then continued for a further 2 hours.

3. Cells are harvested by centrifugation. The cell pellet is resuspended in 10 ml ice cold binding / washing T7-tag buffer (4.29 mM NaH ₂ PO ₄ , 1.47 mM KH ₂ PO ₄ , 2.7 mM KCl, 137 mM NaCl, 1% Tween-20 , 0.02% sodium azide (pH 7.3)).

4. Cells are broken by sonication on ice until the suspension no longer exhibits viscosity.

5. Cell debris is removed by centrifugation (39,000 g, 20 minutes). The supernatant is filtered through a 0.45 µm membrane filter.

6. Load cell extract on a T7-tag antibody agarose (Novagen) column pre-equilibrated with T7-tag binding / wash buffer. The column is then washed with the same buffer to eliminate non-specific bound proteins.

7. The target protein is eluted using an elution buffer (0.1 M citric acid (pH 2.2)).

B. Method using FLAG peptide tag (Sigma) binding of an FLAG peptide tag to an anti-FLAG ™ antibody (Sigma)

The FLAG ™ peptide tag (from Sigma) is an 8 amino acid peptide that is used to purify proteins as " an epitope tag together with an antibody against the epitope. Specifically, a protein is provided having an N-terminal FLAG labeling peptide which is then captured on an anti-FLAG antibody column. The FLAG peptide is used in elution. See P.J. Woodring and J.C. Garnison, Expression, Purification and Regulation of Two Isoforms of Inositol 1,4,5-Trisphosphate 3-Kinase. The Journal of Biological Chemistry 272: 30447-30454,1997.

The following protocol briefly illustrates an example of this process, which may be appropriately modified.

1. Cells (host: B31 cells (Rat-1 fibroblast cell line), vector: pDouble-Trouble (pDT) mammalian expression vector), which express expression of the target protein, are harvested by centrifugation and homogenization in 8 ml hypotonic lysis buffer which it contains protease inhibitors (10 µg / ml calpain inhibitors I and II, 100 µg / ml Pefabloc, 2.5 µg / ml leupeptin, 2 µg / ml aprotinin, 2 µg / ml bacitracin and 20 µg / ml benzamidine).

2. Centrifuge (2,000 g) to remove cell debris and nucleic acids and collect supernatant.

3. After the next. by centrifugation, the cell extract thus obtained is applied to a 1 ml FLAG antibody column. The column is washed with 35 ml TBSC (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% (v / v) CHAPS).

4. The target protein is eluted with 5 ml TBSC containing 200 µg / ml FLAG peptide.

C. Protein A and IgG

This method utilizes the binding of staphylococcal protein A (SPA) to the respective IgG antibody. The fusion protein composed of the target protein and SPA is taken up on a Sepharose IgG column. A low pH buffer is used for elution (see B. Nilsson and L. Abrahmsen, Fusion to Staphylococcal Protein A. Methods in Enzymology 185: 144-161, 1990).

The following protocol briefly shows an example of this process, but it can be modified accordingly.

1. Culture of transformed cells (host: E. coli or

5. aureus, vector: pRIT20 or pRIT30 series cultured overnight is added to 25 ml of LB medium (LB medium + 0.1% (w / v) glucose, 250 mg / l ampicillin) and incubated at 37 ° C for 4 hours.

2. Cells are collected by centrifugation (10,000 g, 5 ° C, 20 minutes) and the supernatant is filtered through a 0.45 µm membrane filter.

3. Load the filtrate onto a 5 ml IgG Sepharose column pre-equilibrated with TST (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% (v / v) Tween 20).

4. Wash the column successively twice with 15 ml aliquots of TST and 5 ml mM ammonium acetate.

5. Protein is eluted with 1 ml 0.5 M ammonium acetate (pH 3.3)

D. Protein and monoclonal antibody

There is also a known method for purifying the cyclic nucleotide closure channel (CNG), a protein that occurs in bovine retina, using the monoclonal antibody PMc 6E7 (N-terminal domain of α subunit: 63 kDa polypeptide) (see RS Molday and LL Molday, Purification) , Characterization, and Reconstitution of Cyclic Nucleotide-Gated Channels. Methods in Enzymology 294: 246-260, 1999). Sepharose 2B (Pharmacia), which carries a monoclonal antibody fixed thereon, was used as the adsorbent. Competitive peptide 6E7, a peptide that competitively binds to a monoclonal antibody and has the amino acid sequence: Ser-Asn-Lys-Glu-Gln-Glu-Pro-Lys-Glu-Lys-Lys-, is used to elute the target protein. Lys-Lys-Lys. This combination is also suitable for use in the present invention.

The following protocol shows an example of a method for purifying a protein derived from bovine retina using a monoclonal antibody.

1. The outer rod segment (ROS) fraction was collected from bovine retina homogenate by centrifugation in a 30 to 50% sucrose density gradient (20 mM Tris-acetate (pH 7.4), 10 mM glucose, 1 mM MgCl 2, 82500 g at 4 ° C for 45 minutes).

2. The ROS fraction was diluted in 5 volumes of homogenization buffer (20% (w / v) sucrose, 20 mM Tris-acetate (pH 7.4), 10 mM glucose, 1 mM MgCl ₂ ) and centrifuged (20000 g). at 4 ° C, 20 min).

3. The ROS pellet was resuspended in 8 ml homogenization buffer to prepare crude ROS extract.

4. ROS was resuspended in 10 volumes of hypotonic lysis buffer (10 mM HEPES-KOH (pH 7.4), 1 mM EDTA, 1 mM DTT) and centrifuged (2000 g, 10 minutes). j

5. The membrane pellet was resuspended in the same buffer and the wash was repeated twice.

6. The pellet was resuspended in 10 mM HEPES-KOH (pH 7.4).

7. ROS membranes were added to CHAPS (3- (3 (cholamidopropyl) dimethylamino) -1-propanesulfonate) solubilization buffer (10 mM HEPES-KOH (pH 7.4), 10 mM CaCl ₂ , 0.15 M KCl, 18 mM CHAPS, 2 mg / ml azolectin (soybean phosphatidylcholine type IV-S, Sigma) protease inhibitor (0.1 mM diisopropylfluorophosphate, 5 μς / ΐ aproteinin, μς / ΐη leupeptin, 2 μρ / π pepstatin or 20 μΜ Pefabloc SC) and stirred slowly.

8. Centrifuge (27000 g, 4 ° C, 30 minutes) was performed to eliminate cell debris.

9. 20 ml of solubilized ROS membranes were loaded onto a column containing Sepharose 2B with fixed PMc 6E7 (antibody). The column was washed with 10 volumes of CHAPS column buffer (10 mM HEPES-KOH (pH 7.4), 1 mM CaCl ₂ , 0.15 M KCl, 12 mM CHAPS, 2 mg / ml azolectin).

10. The target protein was eluted with CHAPS column buffer containing 0.1 mg / ml 6E7 competitive peptide.

(5-3) A method using protein binding to a protein or peptide fragment

A. Strep-Tag and streptavidin

A method for affinity purification of Strep-tag-labeled proteins, an oligopeptide that exhibits affinity for streptavidin, is known (see, for example, A. Skerra and TG Schmidt, The Use of the Strep-Tag and Streptavidin for Detection and Purification of Recombinant Proteins. Methods in Enzymology 326: 271-311 (2000), BioTechniques 28: 338-344 (2000)). This combination is also suitable for use in the present invention.

This method utilizes the Strep-tag binding to streptavidin, using a Strep-tag, e.g. consisting of Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly or Asn-Trp-Ser-His-Pro-Gln -Phe-Glu-Lys (Strep-tag II). A Strep-tagged protein such as DHFR (dihydrofolate reductase) is synthesized in a cell-free system and then purified by adsorption to fixed streptavidin or Strep Tactin. Destiobiotin is used as eluent.

The following protocol shows an example of a method of producing a Strep-tag with a labeled protein component using Strep-tag and streptavidin. There are a number of variations of this method, and so a suitable variation can be easily selected.

1. A culture of transformed E. coli cells (using the pASK-IBA vector) grown overnight was added to 2 L of fresh LB medium (containing ampicillin at a final concentration of 100 µg / ml) and incubated until OD ₅₀ = 0, 5 with shaking (200 rpm) at 22 ° C.

2. 200 μΐ of a 2 mg / ml solution of anhydrotetracycline dimethylformamide (DMF) was added to induce gene expression. The incubation was then continued for a further 3 hours.

3. Cells were harvested by centrifugation (4200 g, 4 ° C, 12 minutes). Then, the cells were resuspended in 20 ml of buffer P (100 mM Tris-Cl (pH 8.0), 500 mM sucrose, 1 mM Na ₂ EDTA) and incubated on ice for 30 minutes.

4. Spheroplasts were centrifuged by centrifugation (27000 g, 4 ° C, 15 minutes).

5. The periplasmic fraction thus obtained was dialyzed overnight against 2 L of buffer (100 mM Tris-Cl (pH 8.0), 1 mM Na 2 EDTA) overnight.

6. StrepTactin-Sepharose column was equilibrated with buffer W. The protein solution was applied to the column using a peristaltic pump, UV detector (A280) and fraction collector system.

7. Wash column with W buffer until A280 reached baseline.

8. The target protein was eluted with buffer W containing 2.5 mM destiobiotin.

B. S-peptide and S-protein

It is known that the protein fragment of ribonuclease S (S-protein) binds strongly, but reversibly, to its S-peptide fragment (S-peptide). The protein can also be purified using this linkage. In particular, an S-labeled protein (i.e. a protein having an S-peptide bound thereto) can be captured on agarose having an S-protein bound. Elution is then effected by cleaving the binding between the S-tag (S-peptide) and the S-protein, for example using a solution of 3M guanidinium thiocyanate, -0.2 M potassium citrate buffer, pH 2.3 M MgCl 2 (see RT Raines, M. McCormick , TRV Oosbree, RC Mierendorf, The S-Tag Fusion System for Protein Purification, Methods in Enzymology 326: 362-376, 2000).

The following protocol briefly shows an example of such a procedure, which may optionally be modified.

1. Add 2 ml S-protein agarose slurry (Novagen) to extract the cells expressing the target protein (host: bacteria, insect or mammalian cells, vector: pET, pBAC (Novagen)) and mix thoroughly. at room temperature for 20 minutes.

2. Centrifuge (500 g, 10 minutes) and discard the supernatant.

3. S-protein agarose having bound target protein is resuspended in binding / wash buffer (20 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 0.1% (v / v) Triton X- 100).

4. After centrifugation (500 g, 10 minutes), supernatant is discarded, eliminating non-specific bound proteins.

5. Resuspend the S-protein agarose cartridge in 1.5 volume of elution buffer (binding / wash buffer + 3 M guanidinium thiocyanate, 0.2 M potassium citrate (pH 2) or 3 M MgCl ₂ ).

6. Incubate at room temperature for 10 minutes with occasional stirring to maintain a suspended state.

7. After centrifugation, the target protein is eluted.

C. Calmodulin-binding protein (CBP) and calmodulin (CaM)

In this procedure, the protein is purified by interaction between calmodulin binding protein (CBP) and calmodulin (see P. Vaillancourt, Chao-Feng Zheng, DQ Hoang, and L. Breister, Affinity Purification of Recombinant Proteins Eused to Calmodulin or to Calmodulin-Binding Peptides Methods in Enzymology 326: 340-362 (2000)). This combination is also suitable for use in the present invention.

First, a CBP-fusion protein is prepared which consists of a protein component and CBP bound thereto. It is then purified by adsorption using a CaM affinity resin based on Sepharose 4B or other commercially available CaM resins. The elution is carried out with EGTA, which is capable of chelating with Ca ²⁺ .

The following protocol shows an example of a method of preparing a CBP-fusion protein using CBP and CaM. A series is known. variations of this method from which a suitable variation can be selected.

1. 20 ml of transformed cell culture (pCA series constructed on pET-11 as a vector) grown overnight was added to 1 liter of LB medium containing 50 µg / ml ampicillin or carbenicillin. The medium was incubated until OD 50 = 0.6-10. After adding IPTG to a final concentration of 1 mM, incubation is continued for an additional 3-5 hours with shaking.

2. Cells were harvested by centrifugation. The cell pellet was resuspended in buffer A (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM 2-mercaptoethanol, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaCl ₂ ) containing 0.2. mg / ml lysozyme. Cells were disrupted by sonication.

3. The cell lysate was centrifuged (25000 g, 15 minutes) and the supernatant was collected.

4. 10 ml CaM-Sepharose resin was equilibrated with buffer A.

5. The CaM-Sepharose resin was mixed with the cell lysate and gently mixed for 1 hour. It was then centrifuged at low

Also speeds, thereby removing the fill and thereby eliminating non-specific bound material.

6. Wash with 40 ml of Buffer A. Resuspend in 20 to 30 ml of Buffer A and then load onto the column. Wash sequentially with 5 column bed volumes of buffer A and buffer B (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM 2-mercaptoethanol, 1 mM magnesium acetate, 1 mM imidazole, 0.1 mM CaCl ₂ ) until the A _{2 S} q UV detector reached the baseline.

7. The target protein was eluted with buffer B containing 2 mM EGTA.

D. HSA and ABP

A method for protein purification has been described using the binding of human serum albumin (HAS) to a serum albumin binding affinity peptide (ABP) (see T. Graslund, J. Nilsson, AM Lindberg, M. Uhlen, and Per-Ake Nygren, Production of Thermostable DNA). Polymerase by Site-Specific Cleavage of Heat-Eluted Affinity Fusion Protein. Protein Expression and Purification 9: 125-132, 1997). The method involves expressing the target protein as a serum albumin-binding affinity peptide (ABP) fusion protein, capturing on an HSA-Sepharose column and then eluting with a low pH buffer. This combination is also suitable for the present invention. The following protocol briefly illustrates this method of protein purification.

1. 5 ml of transformed cell culture (host: E. coli, vector: pET-21a (Novagen) grown overnight) was added to 500 ml TSB + YE medium (30 g / l tryptic soy broth, 5 g / l yeast extract, 100 mg / l ampicillin, 34 mg / l chloramphenicol), followed by incubation with shaking until OD 90 ⁼ 0.8 to 1.5.

2. 1 mM (final concentration) isopropyl-β-D-thiogalactoside was added to induce expression of the fusion protein and incubation continued for an additional 3-5 hours.

3. Cells were harvested by centrifugation. The cell pellet was resuspended in TST (50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 0.05% Tween 20, 1 mM EDTA) and cells were disrupted by sonication.

4. The cell suspension was centrifuged (20,000 g, 30 minutes). The supernatant thus obtained was filtered through a 1.2 µd hydro38 filter.

5. The cell extract was applied to an HSA-Sepharose column.

6. The target protein was eluted with 0.5 M Hac (pH 2.8).

(5-4) Procedures based on the use of protein binding to a specific low molecular weight compound, such as an amino acid, DNA, dye, vitamin or lectin

A. Glutathione-S-transferase (GST) and glutathione

In general, a protein purification method that utilizes an interaction between GST and glutathione is known, known as the "GST pull down method" (see, e.g., Tanpakushitsu Jikken Noto (Protein Experiment Note) (I), on and after p.162, Ch. 5:

1. GST-Yugo Tanpakusthitu no Hatugen to Seisei (Expression and Purification of GST-fusion Protein), Suetake, D. B. Smith, Generating Fusion to Glutathione S-transferase for Protein Studies. Methods in Enzymology 326: 254-270, (2000). This combination is also suitable for use in the present invention.

The method involves preparing a fusion protein that is composed of a target protein with GST and adsorption to glutathione-agarose, which is used as the adsorbent. Reduced glutathione is used as eluent. Where the GST-fusion protein is prepared by genetic engineering techniques, the use of a host organism such as E. coli, Saccharomyces cerevisiae or Saccharomyces pombe is known.

The following protocol shows an example of a method for preparing a GST fusion protein using GST and glutathione. Many variations of this method, from which a suitable version can be selected, are known.

1. Dilute 100 ml of a culture of transformed cells grown overnight to 1 liter of medium containing 100 gg / ml ampicillin followed by incubation.

2. Cells are harvested by centrifugation (5000 g). The cell pellet is resuspended in 20 ml of ice-cold PBS containing a reducing agent such as 1-5 mM dithiothreitol (DTT) or 0.1% 2-mercaptoethanol.

3. Suspended cells are gently sonicated on ice to maintain. The sonication is set to a degree where the solution becomes matt gray in approximately 5 minutes.

4. Add Triton X-100 to a final 1% concentration and centrifuge (10000 g, 4 ° C, 5 minutes). The supernatant is transferred to a 50 ml tube. Add 1 ml of pre-swollen 50% glutathioneagarose beads and incubate at 4 ° C for 30 minutes with occasional inversion.

5. The beads are collected by centrifugation (500 g, 30 minutes) and washed three times with a 50 ml aliquot of ice cold PBS.

6. Elute the fusion protein by gently mixing beads of equal volume with freshly prepared 50 mM Tris-HCl (pH 8) at room temperature for 5 minutes.

7. Remove supernatant by centrifugation (500 g, 30 seconds), add glycerol to a final concentration of 10% and store aliquots at -80 ° C.

B. Protein and dye as ligand;

It is described that native protein originating from Zymononas mobilis shows affinity for specific dyes such as

C.I. 17908, Reactive Red 8 and C.I. Reactive Blue 187 (CI Reactive Blue 187) and that this protein can be purified by utilizing the following properties (RK Scopes and K. Griffiths-Smith, The (Differential and Dye-Ligand Chromatography with Affinity Elution for Enzyme Purification: 6-Phosphogluconate Dehydratase) from Zymononas mobilis. Analytical Bio40 chemistry 136: 530-534, 1984) A particular target protein can be purified by labeling the target protein with all or part of the protein, adsorption to an Sepharose affinity column and using said dye as ligand and finally eluting. The following protocol shows a method of purifying a Z. mobilis protein.

1. The starting material is a liquid culture of Z. mobilis from which the cell extract is prepared using extraction buffer (20 mM K-Mes (pH 6.5), 30 mM NaCl, 5 mM MgCl ₂ , 0.5 mM ferrous ammonium sulfate, 10 mM K-Mes). mM β-mercaptoethanol).

2. The cell extract was sequentially loaded onto a CL-4B column (Pharmacia) containing a dye bound to Sepharose: Scarlet MXG (C.I. 17908, Reactive Red 8) -Sepharose and Blue HE-G (C.I. Reactive Blue 187) -Sepharose.

3. Wash the columns with 100 ml extraction buffer.

4. Only the Blue-HE-G column, not the Scarlet MX-G column, is washed with an extraction buffer containing 20 mM Na ₂ SO ₄ .

5. The target protein originating from Z. mobilis is eluted with 20 mM DL-α-glycerophosphate.

C. Biotin-Avidin Binding Procedure

A method of protein purification using specific binding of biotin to avidin has long been known (see, for example, JD Alche and H. Dickinson, Affinity Chromatographic Purification of Antibodies to and Biotinylated Eusion Protein Expression in Escherichia coli. Protein Expression and Purification 12: 138-143, 1998 ). The method comprises preparing a fusion protein composed of biotin and a target protein using a sequence (122 amino acids) that is biotinylated in a host (e.g. E. coli), capturing the fusion protein on an avidin-coupled column (e.g. avLin soft release avidin resin ( Promega), and then competitive elution with biotin. This combination is also suitable for use in the present invention.

1. To a culture of transformed cells (host: E. coli, vector: PinPoint Xa-2 (Promega)) grown overnight, 1 mM IPTG (i.e. final concentration) is added to induce gene expression and incubated for an additional 5 hours. If the expressed protein is present inside the cells, proceed as follows:

2. Cells are lysed by adding 10 ml of buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl, 0.1 mM PMSE, 1 mg / ml lysozyme) for each gram of cell pellet.

3. Centrifuge (18000 g, 15 minutes) to collect the precipitate containing the target protein and resuspend in buffer (50 mM Tris-HCl (pH 8), 10 mM EDTA, 50 mM NaCl, 0.5% Triton X-100 , 0.1 mM PMSE), followed by two washes.

4. After centrifugation (18000 g, 15 minutes), the pellet thus obtained is resuspended in solubilization buffer (50 mM Tris-HCl (pH 8), 10 mM EDTA, 50 mM NaCl, 0.5% Triton X-100, 0.1 mM PMSF, 6 M guanidine-HCl).

5. Load cell extract onto a 3 ml SoftLink Soft Release Avidin Resin (Promega) column pre-equilibrated with 30 ml of blubble buffer. Wash with 60 ml of solubilization buffer.

6. The biotinylated target protein is purified by a solubilizing eluent containing 5 mM biotin.

(5-5) A procedure that uses protein binding to a carbohydrate

Using this method, a protein is purified using an interaction between a carbohydrate-binding carbohydrate protein. For example, it is known to use a protein that binds maltose (MBP) with amylose (D. Sachdev and J.M. Chirgwin, Fusions to MaltoseBinding Protein: Control of Folding and Solubility in Protein Purification. Methods in Enzymology 326: 312-321 (2000)). In addition, it is expected that interactions between β-galactose binding proteins, such as galactine with β-galactose, can be utilized. These combinations are also suitable for use in the present invention.

A. Maltose and amylose binding protein

In a method wherein the fusion protein with the maltose binding protein is adsorbed using a resin having fixed amylose and using maltose as eluent. The following protocol shows an example of a method for preparing a fusion protein with a protein that binds maltose. A number of variations of this method are known from which a suitable method can be selected.

1. 2 ml culture of transformed cells (F. coli / pMAL-c2 vector (New England Biolabs)) grown overnight were added to 225 ml LBD medium (LB medium containing 0.2% glucose) containing 100 µg / ml ampicillin and incubated at 37 ° C with shaking until an OD of ₆₀ o = 0.5.

2. 0.3 mM isopropyl-β-thiogalactopyranoside (IPTG) was added to induce gene expression and incubation was continued for another 2-3 hours at 30 ° C.

3. Cells were harvested by centrifugation (6800 g, 5 minutes). The cell pellet was resuspended in 10 ml column buffer (20 mM Tris (pH 7.4), 200 mM NaCl, 1 mM EDTA, 0.02% Tween 80) and frozen at -20 ° C overnight.

4. The frozen cells were thawed in ice-water and diluted into 10 ml column buffer. Then, the cells were broken by sonication (intensity: 75% of maximum degree).

5. After centrifugation (20000 g, 4 ° C, 15 min), the supernatant was diluted with 10 ml column buffer (crude extract).

6. The crude extract was applied to a 10 ml amylose resin column.

7. Wash the column several times with 20 to 30 ml aliquots of column buffer. The target protein was then eluted with a column buffer containing 10 mM maltose.

B. Chitin and chitin binding domain (CBD)

This method utilizes chitin binding to a chitin-binding domain (CBD) for protein purification. In particular, a fusion protein composed of a chitin binding protein (CBD) that is bound to a target protein via an intein (ie, an artificial binder that induces self-cleavage activity engineered protein splicing elements) is then expressed and then adsorbed using chitin affinity. columns (New England Biolabs). On elution, the binding between the internal and target protein is cleaved by a reducing agent such as DTT, β-mercaptoethanol or cysteine (see, for example, Chung-Mo Park, Jae-Yoon Shim, Song-Sook Yang, Jeong-GuKang, Jeong-Il Kim) , Z. Luka, and Pill-Soon Song, Chromophore-Apoprotein Interactions in Synechocystits, PCC6803 Phytochrome Cphl. Biochemistry 39: 6349-6356, 2000). When applying this method to the protein components of the present invention, the protein components are labeled with an adhering agent other than CBD-intein, and the adhesive tag is used to separate the protein components from the target protein generated in the in vitro synthesis system.

The following protocol briefly shows an example of such a method for protein purification. A number of variations of this method are known from which a suitable method can be selected.

1. 3 ml culture of transformed cells (host: E. coli, vector: pTYB2 (New England Biolabs)) grown overnight were added to 250 ml of RB medium (0.5% yeast extract, 1% trypton, 0.5% NaCl). 0.2% glucose (pH 7.5)) and incubated at 30 ° C until an OD of ₆ ° C = 0.6.

2. 1 mM IPTG (final concentration) was added to induce expression of the fusion protein and incubation continued for a further 14-16 hours at 20 ° C.

3. Cells were harvested by centrifugation (5000 g, 5 minutes).

The cell pellet was resuspended in lysis buffer (Tris-HCl (pH 8.0), 500 mM NaCl, 0.1% Triton X-100, 1 mM EDTA) while cooling on ice. Cell disruption was performed by sonication.

4. The supernatant was collected by centrifugation (100000 g, 30 minutes) and filtered through a 0.2 mm membrane filter.

5. To 20 ml of cell extract was added 20 μΐ of 2 mM DMSO. Then, the extract was further incubated on ice for 1 hour and plated on a chitin affinity column.

6. Wash the column with buffer (20 mM Tris-HCl (pH 8.0), 150 mM)

NaCl, 1 mM EDTA, 0.1% Triton X-100). It was incubated with a buffer containing 1 mM DTT (final concentration) at 4 ° C overnight to induce intrinsic self-cleavage. Thereafter, the target protein thus released was isolated.

(5-6) A method that uses binding of a protein or a petid fragment to an ion exchange resin

A. Poly-Arg and ion exchange resin

In this method, the protein is purified using the phenomenon that the target protein designated Poly-Arg, which is positively charged, is adsorbed onto a cation exchange resin (e.g. an SP-TSK HPLC column). Elution is accomplished by controlling the ionic strength of the solution (J.

C. Smith, R. B. Derbyshire, E. Cook, L. Dunthorne, J. Viney, S. J. Brewer, H. M. Sassenfeld, and L. D. Bell, Chemical Synthesis and Cloning of a Poly (Arginine) -Coding Gene Fragment Designed to Aid Polypeptide Purification. Gene 32: 321-327 (1984).

The following protocol shows an example of the method described. A number of variations of this method are known from which a suitable method can be selected.

1. 6 ml of transformed cell culture (host !: E. coli, vector: pWT221) grown overnight was added to 300 ml of M9 medium containing 100 µg / ml ampicillin and incubated with shaking at 37 ° C until OD ₅₀ o 0.4

2. A solution of IAA (20 mg / ml in ethanol) was added to a final concentration of 20 µg / ml.

3. 0.1% Polymin P (final concentration) was added and cells were harvested by centrifugation.

4. The cell pellet was lysed in buffer (40 mM Tris-acetate (pH 5.5), 5 M urea) and dialyzed against the same buffer.

5. 0.1 ml of cell extract was loaded onto an SP-TSK HPLC column and the column was washed with the same buffer.

í · ^{1 1}

6. The target protein was eluted with buffer (40 mM PIPES (pH 6.0), 5 M urea) with a NaCl concentration gradient (100 mM to 350 mM).

(5-7) A method using beads

Commercially available magnetic beads (Dynabeads ^Tr?, Dynal, Norway) having a uniform particle size which consist of a polymer core comprising a uniform dispersed magnetizable material (e.g. YFE Fe3O ₂ O 3 and ₄₎ and a shell of hydro46 philic polymer. By binding various antibodies to the surface of these beads, the beads can bind to cells and proteins. When a strong magnet (MPC) is then approached, the magnetic beads are magnetized and magnetically attracted. When the magnet is removed, the beads are demagrietized and dispersed again. Due to these properties, magnetic beads have also been used, for example, to purify cells and proteins. For example, isolation of B cells from peripheral blood using magnetic polystyrene beads (DYNAL) coated with CD19 antibody has been described (Kanegasaki, S. et al., J. Biochem. 117: 758-765 (1995)). Magnetic beads can also be used in the present invention to label the protein components that make up the reaction system and then remove them magnetically from the reaction system. Thus, this combination of magnetic beads and magnet also falls within the category of substances interacting with each other within the meaning of the present invention.

The present invention is further illustrated by the following examples. However, it is to be understood that the invention is not limited to these examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of E. coli ribosomes and extraction of S100

300 g of E. coli A19 cells (harvested in the middle of the log phase) were ground with alumina (alumina). Crushed cells were resuspended in buffer A (10 mM Hepes-KOH (pH 7.6), 10 mM MgCl 2, 50 mM KCl, 1 mM DTT) and alumina and cell debris were removed by centrifugation at 30,000 g for 1 hour at 4 ° C. DNase (deoxybononuclease) was added to the supernatant to a final concentration of 1 µg / ml, followed by centrifugation at 100,000 g for 4 hours at 4 ° C. The supernatant fraction thus obtained was called S100. The pellet was resuspended in buffer A and the resulting suspension was designated as crude ribosome extract. from this crude ribosome extract, a tightly coupled ribosome fraction of 6 to 36% was obtained in a sucrose density gradient. This fraction of tightly coupled ribosomes was centrifuged at 100,000 g and the pellets were resuspended in ribosome buffer (20 mM Hepes-KOH (pH 7.6), 6 mM MgOAc, 30 mM NH ₄ Cl, 7 mM β-mercaptoethanol) to prepare tightly coupled ribosomes. Fig. 1 shows the ribosome fractions in a sucrose density gradient.

Example 2

Construction of a plasmid for expression of initiation, elongation and termination factors

Using the E. coli A19 genome as a template, the gene sequence encoding the EF-Tu gene was amplified by PCR to obtain a DNA fragment having an EcoRI recognition sequence at the 5'-end and a BglII recognition sequence at the 3'-end. The DNA fragments thus obtained were inserted into plasmid pQE60 (QIAGEN), which had previously been digested with BcoRI and BglII. This constructed an overexpression vector of EF-Tu fused to His tag at the C-terminus. Further, E. coli BL21 / pREP4 was transformed with this vector. Vectors to express other elongation factors, initiation factors, and termination factors were constructed in the same manner. Table 1 gives an overview of the vectors and restriction enzymes used and His tag sites.

Example 3

Construction of plasmids for the expression of aminoacyl-tRNA synthetase (ARS) and methionine-tRNA formylase (MTF).

Using the E. coli A19 genome as a template, the gene sequence encoding alanyl-tRNA synthetase was amplified by PCR to produce a DNA fragment having the SphI recognition sequence at the 5'-end and a HindIII recognition sequence at the 3'-end. The DNA fragments thus obtained were inserted into plasmid pQE30 (QIAGEN), which had previously been digested with SphI and HindIII. This constructed an overexpression vector of alanyl-tRNA synthetase fused to the His tag at the N-terminus. Further, E. coli BL21 / pREP4 was transformed with this vector. Vectors to express additional ARS and MTF were constructed in the same manner. Table 1 gives an overview of the vectors and restriction enzymes used and His tag sites. The plasmids obtained were transformed into E. coli strain BL21 / pREP4 (pQE series) or BL21 / DE3 (pET series).

Enzyme or vector N-terminal C-terminal Umiel factor R.E. R.E. his- Alarsas pQE30 SphI Hind III N ArgRS pET16b Nde BamHI N AsnRS pQE30 BamHI Hind III N AspRS pET21a Nde XhoI C CysRS pET21a Nde XhoI C GlnRS pET21a Nde XhoI C GluR pET21a Nde XhoI C GlyRS pET21a Nde XhoI C HisRS pET21a Nde XhoI C IleRS pET21a Nde Hind III C leurs pET21a XbaI XhoI C LysRS pET21a Nde XhoI C metrs pET21a XbaI XhoI C PheRS pQE30 SphI Hind III N prorsa pET21a Nde XhoI No. SERRS pET21a XbaI XhoI C ThrRS pQE30 BamHI Hind III N TrpRS pET21a Nde XhoI C TyrRS pET21a Nde XhoI C ValRS pET21a XbaI Noti . C MTF pET21a Nde XhoI C IF1 pQE30 BamHI Hind III N IF2 pQE30 BamHI Hind III N IF3 pQE30 BamHI Hind III N

EF-G pQE60 Mun BglII C EF-Tu pQE60 EcoRI BglII C EF-Ts pQE60 ncol BamHI C RF1 pQE60 BamHI Hind III C

Znamenáοζη .: RE stands for restriction enzyme.

Example 4

Construction of a plasmid for expression of T7 RNA polymerase

Using the T7 phage genome as a template, a gene sequence that encodes a T7 RNA polymerase was amplified by PCR to produce a DNA fragment having a BamHI recognition sequence at the 5'-end and a PstI recognition sequence at the 3'-end. The DNA fragments thus obtained were inserted into plasmid pQE30 (QIAGEN), which had previously been digested with BamHI and PstI. This constructed an overexpression vector of the T7 RNA polymerase fused to the His tag at the N-terminus. Further, E. coli BL21 / pREP4 was transformed with this vector.

Example 5

Construction of plasmids for expression of nucleoside diphosphate kinase (NDK) and other enzymes

Using the E. coli A19 genome as a template, the gene sequence encoding NDK was amplified by PCR to produce a DNA fragment having a BamHI recognition sequence at the 5'-end and a HindIII recognition sequence at the 3'-end. The DNA fragments thus obtained were inserted into plasmid pQE30 (QIAGEN), which had previously been digested with BamHI and HindIII. This constructed a vector for overexpression of NDK, which is fused to the His tag at the N-terminus. Further, E. coli BL21 / pREP4 was transformed with this vector. Vectors to express the other enzymes listed in sections (4-1) and (4-2) were constructed in the same manner.

Example 6

Overexpression and purification of initiation factors, elongation factors, and termination factors

For expression (overexpression) of His-tagged EF-Tu (EF-TU *), the BL21 / pREP4 cells obtained in Example 2 grown in 6 L of LB medium were transformed to an optical density (OD ₆ µm) of 0.7. IPTG (isopropyl-1-thio-β-D-galactoside) was added to the culture to a final concentration of 0.1 mM and incubation was continued for another 4 hours at 37 ° C. Cells were harvested by centrifugation, resuspended in resuspension buffer (50 mM Hepes-KOH (pH 7.6), 1 M NH ₄ Cl, 10 mM MgCl ₂ , 0.3 mg / ml lysozyme, 0.1% Triton X-100 , 0.2 mM PMSF (phenylmethanesulfonyl fluoride), 6 mM β-mercaptoethanol) and broken by sonication. Cell debris was removed by centrifugation at 100,000 g for 1 hour at 4 ° C. The supernatant obtained was loaded onto a 10 ml pre-packed Ni ²⁺ Hi-Trap chelation column (Pharmacia) and the column was then washed with 100 ml HT buffer (50 mM Hepes-KOH (pH 7.6), 1M NH ₄ Cl, 10 mM MgCl ₂ ) containing 10 mM imidazole. Then, EF-Tu * was eluted from the column in a linear gradient of imidazole concentration (10-400 mM) in HT buffer. The EF-Tu * fractions thus purified were collected and dialyzed against buffer stock (50 mM Hepes-KOH (pH 7.6), 100 mM KCl, 10 mM MgCl ₂ , 30% glycerol). The concentration of purified EF-Tu * was determined based on a standard curve), which was prepared using a protein assay kit (BioRad) using BSA (bovine serum albumin) as a standard. The obtained EF-Tu * was rapidly frozen in 1 ml aliquots in liquid nitrogen and then stored at -80 ° C. Other His-labeled elongation factors, initiation factors and termination factors were purified in the same manner. Fig. 2 shows 12% SDS-PAGE profiles of His-labeled factors (after Cooomassie staining with brilliant blue).

Activities and optimal concentrations of His-tagged initiation factors (IF1, IF2, and IF3: means His-tagged) were measured using DHFR mRNA in vitro translation system (see below, Example 17). Using a system containing all IF1 *, IF2 *, and IF3 * as a positive control, incubation was performed for 30 minutes in systems lacking the appropriate initiation factors, and then relative DHFR activities were compared (Fig. 3A). In particular, the activity of His-labeled initiation factors was determined by determining the activity of the positive control as 100. As a result, it was confirmed that the DHFR yield in each non-IF * system corresponds to 4 or less positive control yield, indicating that all IF1 *, IF2 * and IF3 * have activity. The optimal concentration of His-labeled initiation factors was measured by translation in an in vitro system under constant conditions when the concentration of each initiation factor was varied and the relative activity of the DHFR so formed was measured (Fig. 3B). In FIG. 3B, ·, A and, respectively, represent data for IF1 *, IF2 * and IF3 *.

Example 7

Overexpression and purification of His-tagged ARS and MTF

Transformed BL21 / DE3 cells for expression of His-tagged Ser-tRNA synthetase ("* means" His-tagged) were grown in 2 L of LB medium to an optical density (Οϋβοο) of 0.7. IPTG (isopropyl-1-thio-β-D-galactoside) was added to the culture to a final concentration of 0.1 mM and incubation continued for another 4 hours at 37 ° C. Cells were harvested by centrifugation, resuspended in resuspension buffer (50 mM Hepes-KOH (pH 7.6), 1 M NH ₄ Cl, 10 mM MgCl ₂ , 0.3 mg / ml lysozyme, 0.1% Triton X-100, 0.2 mM PMSF (phenylmethanesulfonyl fluoride), 6 mM β-mercaptoethanol) and broken by sonication. Cell debris was removed by centrifugation at 100,000 g for 1 hour at 4 ° C. The obtained supernatant was loaded with 52 salts of 10 ml pre-packed Ni ²⁺ Hi-Trap chelation column (Pharmacia) and the column was then washed with 100 ml HT buffer (50 mM Hepes-KOH (pH 7.6), 1M NH ₄ Cl, 10 mM MgCl ₂ ) containing 10 mM imidazole. Then, Ser-tRNA synthetase * was eluted from the column in a linear gradient of imidazole concentration (10-400 mM) in HT buffer. The Ser-tRNA synthetase * fractions thus purified were collected and dialyzed against buffer stock (50 mM Hepes-KOH (pH 7.6), 100 mM KCl, 10 mM MgCl ₂ , 30% glycerol). The concentration of purified Ser-tRNA synthetase * was determined based on a standard curve prepared using a protein assay kit (BioRad) using BSA (bovine serum albumin) as a standard. The obtained Ser-tRNA synthetase * was rapidly frozen in 1 ml aliquots in liquid nitrogen and then stored at -80 ° C. Fig. 5 shows a chromatogram of the Ser-tRNA synthetase * thus prepared. Additional ARS * and MTF were expressed and purified in the same manner. Fig. 6 shows 12% SDS-PAGE profiles of His-labeled factors and enzymes (Cooomassie brilliant blue stained). The two Gly-RS * and Phe-RS * bands observed in FIG. 6 can be explained by the fact that these enzymes were of types a2 and β2. Fig. 6 shows which His-labeled factors and enzymes were obtained and in what purity.

Example 8

Overexpression and purification of His-tagged T7 RNA polymerase

Transformed BL21 / pREP4 cells for expression of His-tagged T7 RNA polymerase ("* means" His-tagged) were grown in 6 L of LB medium to an optical density (OD of ₆ µm) of 0.7. IPTG (isopropyl-1-thio-β-D-galactoside) was added to the culture to a final concentration of 0.1 mM and incubation continued for another 4 hours at 37 ° C. Cells were harvested by centrifugation, resuspended in resuspension buffer (50 mM Hepes-KOH (pH 7.6), 1 M NH ₄ Cl, 10 mM MgCl ₂ , 0.3 mg / ml lysozyme, 0.1% Triton X-100, 0.2 mM PMSF (phenylmethanesulfonyl fluoride), 6 mM β-mercaptoethanol) and broken by sonication.

Cell debris was removed by centrifugation at 100,000 g for 1 hour at 4 ° C. The supernatant obtained was loaded onto a 10 ml pre-packed Ni ²⁺ Hi-Trap chelation column (Pharmacia) and the column was then washed with 100 ml HT buffer (50 mM Hepes-KOH (pH 7.6), 1M NH ₄ Cl, 10 mM MgCl ₂ ) containing 10 mM imidazole. Then, T7 RNA polymerase * was eluted from the column in a linear gradient of imidazole concentration (10-400 mM) in HT buffer. The T7 RNA polymerase * fractions thus purified were collected and dialyzed against buffer stock (50 mM Hepes-KOH (pH 7.6), 100 mM KOI, mM MgCl ₂ , 30% glycerol). The concentration of purified T7 RNA polymerase * was determined based on a standard curve prepared using a protein assay kit (BioRad) using BSA (bovine serum albumin) as a standard. The obtained T7 RNA polymerase * was rapidly frozen in 1 ml aliquots in liquid nitrogen and then stored at -80 ° C. Fig. 12A and 12B show chromatograms of the T7 RNA polymerase * thus prepared.

Example 9

Overexpression and purification of His-tagged NDK and other enzymes

Transformed BL21 / pREP4 cells for expression of His-tagged NDK ("* means" His-tagged) were grown in 2 L of LB medium to an optical density (OD ₆₀ °) of 0.7. He joined the culture

IPTG (isopropyl-1-thio-β-D-galactoside) to a final concentration of 0.1 mM and incubation continued for another 4 hours at 37 ° C. Cells were harvested by centrifugation, resuspended in resuspension buffer (50 mM Hepes-KOH (pH 7.6), 1 M NH ₄ Cl, 10 mM MgCl ₂ , 0.3 mg / ml lysozyme, 0.1% Triton X-100, 0.2 mM PMSF (phenylmethanesulfonyl fluoride), 6 mM β-mercaptoethanol) and broken by sonication. Cell debris was removed by centrifugation at 100,000g for 1 hour at 4 ° C. The obtained supernatant was loaded onto a 10 ml pre-packed Ni ²⁺ Hi-Trap chelation column (Pharmacia) and the column was then washed with 100 ml HT buffer (50 mM Hepes-KOH (pH 7.6), IM).

I

NH 4 Cl, 10 mM MgCl ₂ ) containing 10 mM imidazole. Then

NDK * eluted from the column in a linear gradient of imidazole concentration (10-400 mM) in HT buffer. NDK * fractions thus purified were collected and dialyzed against buffer stock (50 mM Hepes-KOH (pH 7.6), 100 mM KCl, 10 mM MgCl ₂ , 30% glycerol). The concentration of purified NDK * was determined based on a standard curve prepared using a protein assay kit (BioRad) using BSA (bovine serum albumin) as a standard. The obtained NDK * was rapidly frozen in 1 ml aliquots in liquid nitrogen and then set aside at -80 ° C. The other enzymes and factors described in sections (4-1) and (4-2) were obtained in the same manner.

Example 10

DHFR gene construction and mRNA preparation

By adding the HindIII sequence and the BamHI sequence, at the 5'-end and 3'-end as indicated, of the DHFR (dihydrofolate reductase) gene originating from E. coli, this gene was amplified by PCR. The gene contains upstream of the ribosome T7 promoter binding site with an epsilon sequence derived from T7 bacteriophage gene 10 followed by a Shine-Dalgarno (SD) sequence. This DNA fragment was cloned into the plasmid vector pUC18 (Takara Shuzo). After digestion with SmaI, this plasmid was used as a template for run-off transcription or in vitro translation coupled using T7 RNA polymerase. The in vitro transcription reaction was performed at 42 ° C for 3 hours. Reaction mixture (1 mL) containing 40 mM Hepes-KOH (pH 7.8), 20 mM MgCl ₂ , 1 mM spermidine, 5 mM DTT, 2 mM each of ATP, UTP, CTP and GTP, 20 µg Smal-cleaved template plasmid, 50 µg BSA, 1.78 units Ppiase (pyrophosphatase) and 10 µg purified His-tagged T7 RNA polymerase. To stop, EDTA (ethylenedinitrotetraacetic acid) was added to a final concentration of 50 mM. The mRNA thus obtained was extracted with phenol / chloroform and then precipitated with ethanol. It was further purified using the RNA Purification Kit (QIAGEN) according to the manufacturer's instructions.

Example 11

Construction of MFL mRNA

To obtain a template for the AUGUUCUUGUAA MFL mRNA, a DNA sequence (translated into the fMet-Phe-Leu-Stop sequence; i.e., formylmethionine-phenylalanine-leucine-stop codon, hereinafter abbreviated to MFL) was constructed as follows:

Oligonucleotide A: 5'-TAtgttcttgtaac was complementary linked to the second oligonucleotide B: 5'-TCGAgttacaagaaca to form double stranded DNA containing NdeI and XhoI sequences. Then this DNA was cloned into the NdeI and XhoI sites of the plasmid vector pET29a (Novagen). The resulting plasmid was transcribed in the same manner as described for the DHFR gene.

Example 12

Activities of His-labeled aminoacyl-tRNA synthetase

The activities of His-labeled ARS (aminoacyl-tRNA synthetases) were measured as follows. Each reaction mixture (50 μΐ) contained a "polymix buffer (see also translational experiments) that contained 1 mM ATP, 2.8 A ₂ 6 units tRNAmix (Boehringer)> 50 μΜ of each of the labeled amino acids and each of the purified His-labeled ARS . Reactions were performed at 37 ° C and radioactive aminoacyl-tRNA was precipitated on 3MM filters at different times of incubation and washed with cold 5% trichloroacetic acid, and then radioactivity was measured. One unit of activity was defined as the amount of enzyme capable of synthesizing 1 pmol aminoacyl-tRNA per minute.

Table 2 shows the results

Table 2 enzyme concentration (Μς / μΐ) Specific activity (U / pg) Units required for reaction - '(50 μΐ reaction) Alarsas 13 27 94 ArgRS 10 1300 130 AsnRS 30 ND ND AspRS 22 310 130 CysRS 25 500 31 GlnRS 36 330 63 GluR 26 150 94 GlyRS 30 520 250 HisRS 30 1600 31 IleRS 20 63 130 leurs 22 940 190 LysRS 35 580 190 metrs 27 3000 310 PheRS 23 15 63 prorsa 16 120 63 SERRS 17 1000 94 ThrRS 19 200 63 TrpRS 11 600 31 TyrRS 22 1800 31 ValRS 20 1700 156 MTF 12 230 230 ND = not specified the

Example 13

Activities of His-labeled methionyl-tRNA transformylase

The activities of His-tagged MTF (methionyl-tRNA transformylase) were measured as follows ("*" refers to His-tagged enzyme). Each reaction mixture (50 μΐ) contained a 'polymix buffer (see also translation experiments) containing 1 mM ATP, 2.8 A ₂₆₀ tRNAmix units (Boehringer), 50 μΜ [ ³ H] -labeled methionine, 0.5 μg 10-formyl -5,6,7,8-terahydrofolic acid, 3000 MetR and MTF units *. Reactions were carried out at 37 ° C and unformylated methionyl-tRNA was deacylated in a buffer containing 0.175 M CuSCl 2 and 0.5 M Tris-HCl (pH 7.5) for 8 minutes at 30 ° C. Radioactive formyl-methionyl-tRNA was precipitated on 3MM filters at various times of incubation and washed with cold 5% trichloroacetic acid, and then radioactivity was measured. One unit of activity was defined as the amount of enzyme capable of synthesizing 1 pmol of formylmethionyl-tRNA per minute. The results are also shown in Table 2 (last column).

Example 14

Translation Experiments (General)

Translation mixtures (50 µl) were prepared by small modification of the "polymix buffer used in Jelene et al. (1979) and Wagner et al. (1982). The "Polymix" buffer contained 5 mM magnesium acetate, 5 mM potassium phosphate (pH 7.3), 95 mM potassium glutamate, 5 mM ammonium chloride, 0.5 mM calcium chloride, 1 mM spermidine, 8 mM putrescine and 1 mM DTT. The reaction mixture contained 1 mM ATP, 1 mM GTP, 10 mM creatine phosphate, 2.8 Å ₂₈ tRNAmix units, 0.5 µg 10-formyl-5,6,7,8-tetrahydrofolic acid, 1 mM each of the amino acids, and a mixture of factors as described below. In the case of a coupled transcription and translation reaction, 1 mM each of the NTP and 4 mM magnesium acetate were further added to said reaction mixture. The factor-enzyme mixture contained 12 pmol ribosomes, 1 µg IF1 *, 2 µg IF2 *, 0.75 µg IF3 *, 1 µg EF-G *, 2 µg EF-Tu *, 1 µg EF-Ts *, 0.5 pg RF1 *, 0.5 µg RF3 *, 0.5 µg RRF *, 30 to 300 units of each ARS * or MTF, 0.2 µg creatine kinase (CK), 0.15 µg myokinase (MK) and 0.054 µg nucleoside diphosphate kinase (NDK). In addition, for translation-related transcription, 1.78 units of Ppiase and 0.5 µL of T7 RNA polymerase * were added. In these factors and enzymes, labeling is by

His-tag. The reaction mixtures were incubated at 37 ° C for 5 minutes, and then template DNA or RNA was added and the reaction started. The translation was performed at 37 ° C. After completion of the reaction, ribosomes having a high molecular weight were first removed by passing through an ultrafiltration membrane with a size limit of 100 kDa. Then the fraction was filtered through an ultrafiltration membrane applied to a Ni column to remove His-tagged fusion proteins. The component passed through the column is a translation product having a high degree of purity, as evidenced by a single band on SDS-PAGE. The S30 system used in the following experiments was purchased from Promega and translation was performed according to the manufacturer's recommended protocol.

Example 15

Expression of various proteins

After confirming the activity of His-tagged components of the reaction system, an in vitro protein synthesis reaction system was also constructed, which was also used in Example 14, using His-tagged T7 RNA polymerase. Using this system, the synthesis of full-length E. coli DHFR polypeptides, λ-lysozyme, green fluorescent protein (GFP), glutathione transferase (GST), and the T7 gene phage 10 protein was determined and yields of each product were measured. The results are shown in FIG. The system according to the invention has been shown to contain all the components necessary for translation.

Example 16

Synthesis of poly (U) -poly (Phe)

Poly (U) -poly (Phe) was synthesized in the in vitro reaction system as follows. Each reaction mixture contained a polymix buffer containing 1 mM ATP, 1 mM GTP, 10 mM creatine phosphate,

2.8 Α280 ^- tRNAmix units, 1 mM [ ¹⁴ C] -labeled phenylalanine and a mixture of factors. The factor mixture contained 12 pmol ribosomes, 1 µ IF1 *, 2 µ IF2 *, 0.75 µ IF3 *, 1 µ EF-G *, 2 µ EF-Tu *, 1 µg EF-Ts *, 0.5 µg RF1 *, 0.5 µg RF3 *, 0.5 µg RRF *, 60 PheRS * units, 0.2 µg creatine kinase (CK), 0.15 µg myokinase (MK), and 0.054 µg nucleoside diphosphate kinase * (NDK *). Among the factors and enzymes, those marked with a * are indicated with a His-tag. The reaction mixtures were incubated at 37 ° C for 5 minutes and then 5 µg of poly (U) was added and the reaction was started. Poly (Phe) was collected at time intervals in 8 μΐ aliquots and precipitated on 3MM filters with 10% trichloroacetic acid. Aminoacyl-tRNA was deacylated at 85 ° C and washed with 10% trichloroacetic acid followed by measurement of radioactivity. This confirmed the formation of the target product.

In the translation reaction described, optimal concentrations of EF-G *, EF-Tu * and EF-Ts * were determined by determining poly (Phe) yield in the in vitro reaction system under constant conditions but with varying concentrations of each of the elongation factors . In FIG. 7 (A) are the symbols ·, A and the results shown for EF-G *, EF-Tu * and EF-Ts, respectively.

Further, data on the synthesis of poly (Phe) in the described reaction system of the present invention were compared with data for a translation system using the S100 extract. Fig. 7 (B) shows the result. Although in the second system (o) used, the reaction stopped as early as 20 minutes, in the system of the present invention (·) the reaction continued after 40 minutes.

Example 17

Activities of termination and ribosome recycling factors

Termination factor activities (RF1 *, RF3 *, and RRF *, denoted by the name "His-tagged") were measured according to Pavrov et al. (8) with little modification. The reaction mixture (50 μΐ) was prepared from the 'polymix buffer used in translational experiments. Each reaction mixture comprised 1 mM of ATP, 1 mM GTP, 2.8 and ₂ 6o unit tRNAmix, 1 mM of ^[14 0] -labeled phenylalanine and leucine, 50 pmol of formyl-methionyl-tRNA prepared by using ^[35 S] -labelled methionine, and mixture of factors and His-labeled enzymes (as described). The factor mix contained 12 pmol ribosomes, 1 µ IF1 *, 2 µ IF2 *, 0.75 µ IF3 *, 1 µ EF-G *, 2 µ EF-Tu *, 1 µ EF-Ts *, 0.5 µg RF1 *, 0.5 µF RF3 *, 0.5 µF RRF *, 50 PheRS * 300 LeuRS *. RF1 *, RF3 *, and RRF * were individually removed from the reaction mixture depending on the purpose of that reaction mixture. The reaction mixtures were incubated at 37 ° C for 5 minutes then 1 µL of MFL mRNA was added to initiate translation. Samples were then taken from the reaction mixture in 5 μΐ aliquots and each sample ¹ was added to an equal volume of 1 N HCl to stop the reaction. Then 200 μΐ of ethyl acetate was added to elute the tripeptide (fMFL) and the radioactivity was measured on a liquid scintillation counter.

The activities of termination factors RF1 *, RF3 *, and RRF * were measured using an in vitro synthetic mRNA translation system encoding fMet-Phe-Leu-Stop (fMFL). Specifically, the fMFL mRNA was translated in a system containing RF1 *, RF3 * and RRF * (·), a system containing RF1 * and RRF * (·), a system containing RF1 * and RF3 * (A), a system containing RF1 alone ( ), a system containing RF3 * and RRF * (o) and a system containing no termination factor (♦), and yields were measured. The results are shown in FIG. 4th

In FIG. The 4 peptide synthesized in the first cycle corresponds to approximately 1000 cpm on the y-axis. A system containing RF1 *, RF3 *, and RRF * (·) shows a linear increase in fMFL yield over time, indicating that RF1 *, RF3 *, and RRF * have activities comparable to other systems. In a system without RF1 * (o), no peptide synthesis occurred. Ribosome recycling has not occurred in systems without RRF * (A ♦).

Example 18

Synthesis of DHwFR

DHFR containing [ ^3d S] methionine was synthesized using the in vitro translation system of the present invention and further using the S30-based translation system (Promega). Each product was separated on 12% SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) and detected using a BAS-1000 system (Fuji Film) followed by radioactivity measurement. Fig. 8 (A) shows the results of separation on SDS-PAGE.

Further, DHFR activity was measured as follows. In a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 50 μΜ DHF (dihydropholic acid) and 60 μΜ NADPH (reduced nicotinamide adenine dinucleotide phosphate) DHFR was synthesized at 30 ° C and A3 ₄₀ reduction was measured every minute. The results are shown in FIG. 8 (b)

Fig. 9 shows the reaction progress in the in vitro translation system of the present invention and in the S30 system. In the in vitro translation system of the present invention (), the reaction continued even after 120 minutes while the DHFR yield in the S30 system (·) peaked after 20 minutes.

To investigate the energy consumption, nuleoside triphosphate was hydrolyzed in the in vitro system of the present invention in the S30 system. Hydrolysis in each system was monitored as follows and data were compared. Using DHFR templates, translation was performed in a reaction mixture containing [α-P] ATP or GTP at 37 ° C. Samples were taken from each reaction mixture in 2 µΐ aliquots at intervals, each sample being added to 150 µΐ 10% formic acid. Thereafter, the mixture was applied as a spot to thin-layer chromatography (TLC) plates with polyethyleneimine and the reaction products were developed using 0.75 M potassium phosphate buffer (pH 3.75). After air drying, the TLC plates were wrapped in plastic film and subjected to autoradiography. In FIG. 10 shows the results: the result from the system S30 is shown on the left, the result from the system according to the invention is shown on the right. In the S30 system, ATP decreased over time, while in the system of the present invention, the amount of ATP was kept almost constant. To purify the synthesized DHFR, ribosomes having a high molecular weight were first removed by passing through an ultrafiltration membrane with a size limit of 100 kDa. Then, all His-labeled components that form the reaction system were removed by passing the reaction mixture through a Ni column. The reaction mixture prior to passing through the Ni column and the resulting product were analyzed by SDS-PAGE and stained with Coomassie Blue. Fig. 14 shows the results: lane 1 are markers, lane 2 represents the reaction mixture and lane 3 is a product (DHFR), demonstrating that the product was obtained as a single band.

Example 19

Incorporation of valine residue using valyi-suppressor tRNA (non-natural amino acid incorporation model)

In the in vitro translation system of the present invention containing RF2 * ("* means" His-tagged) as a replacement for RF1 * as a termination factor, the DHFR template in which the Asn at position 37 (ATA codon was changed to UAG codon) was translated using chemically synthesized valyi-suppressor tRNA. As a result, a truncated protein ending in residue 37 (Fig. 11, lane 2) was synthesized in the RF1-containing sample, while a weak band corresponding to this truncated protein was observed by the RF1 *-free system (not including RF2) (Fig. 11, lane 3) ). In a system incorporating RF2 *, the protein product was observed at the same position (Figure 11, lane 4) as normal DHFR (Figure 11, lane 1). Based on these results, it was confirmed that the valine residue bound to the suppressor tRNA was incorporated at position 37 in DHFR.

Industrial usability

By labeling the protein components that make up the reaction system, the individual protein components making up the reaction system can be readily purified with certainty, and thus it is possible to construct a reaction system that is not contaminated with any unknown components. Moreover, such a system makes it possible to easily isolate the target protein thus synthesized in high purity.

Although it has already been pointed out that the lipopolysaccharides (LPS) contained in cells and cell extracts exhibit various undesirable effects as endotoxins on the living organism, there are technical problems why it is difficult to separate these LPS from the target peptide products. However, these problems are solved using the in vitro peptide synthesis system of the present invention.

By building a reaction system that does not contain any unknown components, reactions can take a long time, for example 2 hours or more, in a batch system. Moreover, it is at least theoretically possible to increase the volume of the reaction mixture.

By using a flow system, it can be achieved that reactions can take place for a very long time, allowing industrial production and purification of the protein in an in vitro reaction system. Thus, such a process makes it possible to economically produce certain enzymes that have been reluctantly used for medical applications due to their high cost. Thus, by using the present invention, the spectrum of treatment can be extended by administering enzymes that have so far been used as substitution therapy instead of gene therapy that still suffers from the technical problems of gene delivery.

According to the present invention, a system comprising termination factors and a system completely free of termination factors can be assembled and ribosome displays can be easily and selectively prepared. In addition, it is thus possible to more accurately incorporate non-natural amino acid residues into the desired position in the protein.

Conventional cell-free protein synthesis systems that use prokaryotic cell extracts suffer from a number of problems, particularly that mRNA stability is significantly reduced when transcription and translation are not performed simultaneously. In contrast, the mRNA translation reaction can be stably performed in the reaction system of the present invention.

After genome analysis, gene research is then the main direction in molecular biology research. In these circumstances, the present invention, which accelerates gene expression and identification of protein products and thereby facilitates the detection of gene function, contributes significantly to advances in science and technology.

Cited publications:

1) Crowe, J., Dobeli, H., Gentz, R., Hochuli, E., Stuber, D., and Henco, K. (1994). 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression / purification. Methods Mol Biol 31: 371-87.

2) Hochuli, E., Dobeli, H., and Schacher, A. (1987). New metal chelate adsorbent selective for proteins and peptides containing neighboring histidine residues. J Chromatogr 411,177-84

3) Smith, D. B., and Johnson, K. S. (1988). Single-step purification of polypeptides expressed in Escherichia coli as fusion with glutathione S-transfection. Gene 67: 31-40.

list sequences <110> POST GENOME INSTITUTE CO., LTD

<120> Method for preparing peptides using in vitro transcription / translation system <130> NTK01-1399

<140> PCT / JP 01/10682 <141> 2001-12-06 <150> JP 294795/2001 <151> 2001-09-26 <150> JP 227094/2001 <151> 2001-07-27 <150> JP 6910/2001 <151> 2001-01-15 <150> JP 401417/2000 <151> 2000-12-28 <160> 6 <170> Patent version 3.1 <210> 1 <211> 14 <212> PRTs <213> Bovine sp.

<400> 1

Ser Asn Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys

1 5 10 <210> 2 <211> 9 <212> PRTs <213> Artificial sequence

<220>

<223> Streptavidin-binding label

<220>

<221> Various features <222> (D) · (9) <223> Schmidt & Skerra, 1993, "The random peptide library-

assisted engineeration of C-terminal affinity peptide, useful for detection and purification of IgFv fragment function, Protein Eng. 6 (1): 109-122.

<400> 2

Ala Trp His His Gin Phe Gly Gly

5

<210> 3 <211> 9 <212> PRTs <213> Artificial sequence

<220>

<223> Strep-tag tag <220> <221> Various features <222> (1). (9) <223> Schmidt et al., The Strep-tag affinity Streptavidin, J. of Mol. <400> 3 Asn Trp Ser His' Pro Gin Ph 1 5 <210> 4 <211> 12 <212> RNA <213> Artificial sequence <220> <223> RNA sequence on <220> <221> mRNA <222> (1) <22 3> <400> 4

auguucuugu aa <210>

<211> 14 <212> DNA <213> Artificial sequence <220> <223> oligonucleotide And, which be complementary binds to sequence SEQ ID NO: 6 what arises double stranded DNA contain an account

NdeI and XhoI sequences <400> 5 tatgttcttg taac <210> 6 <211> 16 <212> DNA <213> Artificial sequence <220>

<223> oligonucleotide B which be complementary binds to sequence SEQ ID NO: 5 what arises double stranded DNA comprising sequence Ndel and Xhol <400> 6

tcgagttaca agaaca

7T M6 ~ Z.eo? __

Claims (18)

A method for preparing a peptide or peptide derivative using a DNA-to-RNA transcription reaction system and then translation of the generated RNA or in vitro RNA translation reaction system, characterized in that part or all of the protein components that make up the reaction system are labeled by one of a pair The second agent from the pair is used as an adsorbent to capture the labeled protein components after translation. A process for the preparation of a peptide or a peptide derivative according to claim 1, characterized in that a plurality of combinations of a substance used to label part or all of the protein components that make up the reaction system are used with a substance used as an adsorbent to capture the labeled protein components. . A process for the preparation of a peptide or peptide derivative according to claim 1, characterized in that the protein components labeled with one of a pair of substances that adhere to each other are part or all of the factors and enzymes for the transcription or translation reaction. The method of preparing a peptide or peptide derivative according to claim 3, wherein the factors and enzymes for the transcription or translation reaction are selected from the group consisting of initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetase, methionyl-tRNA transformylase. and RNA polymerase. A process for the preparation of a peptide or peptide derivative according to claim 1, characterized in that the protein components labeled with one of a pair of substances that adhere to each other are factors and enzymes for a transcriptional or translational reaction and other enzymes necessary to assemble the reaction system. A process for the preparation of a peptide or peptide derivative according to claim 5, characterized in that the enzymes necessary for assembly of the reaction system, other than the factors and enzymes for the transcription or translation reaction, are selected from the group consisting of enzymes for energy recovery in the reaction system; enzymes for the hydrolysis of inorganic diphosphoric acid formed during the transcription or translation reaction. 7. A method according to claim 1, wherein the reaction system for transcribing DNA into RNA and then the translation of the generated RNA or the in vitro translational RNA reaction system does not contain termination factors. A process for the preparation of a peptide or peptide derivative according to claim 1, characterized in that the pair of mutually adhering substances are substances which interact with each other in affinity chromatography. 9. A method according to claim 8, wherein the combination of substances interacting in affinity chromatography is selected from combinations of substances capable of binding between a protein or peptide fragment and a metal ion, an antigen-antibody binding, a bond. between a protein and a protein or peptide fragment, a binding between the protein and a specific low molecular weight compound selected from the group consisting of amino acids, DNA, dyes, vitamins and lectins, a protein-carbohydrate or protein-peptide fragment binding and ion exchange resin. 10. A method according to claim 9, wherein the combination of substances that form a bond between the protein or peptide fragment and the metal ion is a histidine tag and a nickel complex or cobalt complex. The method of preparing a peptide or peptide derivative according to claim 1, wherein the pair of substances that adhere to each other are magnetically adhering to each other. 12. A kit of protein components for a reaction system for preparing a peptide or peptide derivative by transcribing DNA into RNA and then by translation generated by RNA or by in vitro translation of RNA, characterized in that it contains some or all of the protein components that make up the reaction system and selected from the group consisting of enzymes and factors labeled with one of a pair of adhering substances. A kit of protein components according to claim 12, characterized in that the protein components are selected from the group of factors and enzymes for the transcriptional or translational reaction and other enzymes necessary for the assembly of the reaction system. 14. The protein component set of claim 13, wherein the factors and enzymes for the transcription or translation reaction are selected from the group consisting of initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetase, methionyl-tRNA transformylase, and RNA polymerase. 15. A kit of protein components according to claim 13, wherein the enzymes required for assembly of the reaction system other than the factors and enzymes for the transcription or translation reaction are selected from the group consisting of enzymes for energy recovery in the reaction system and enzymes for hydrolyzing inorganic diphosphoric acid. , which is formed during a transcription or translation reaction. 16. A protein component kit according to claim 12 comprising an adsorbent for capturing the protein components labeled with one of a pair of adhering substances. 17. A protein component kit according to claim 12, comprising a plurality of combinations or pairs of labeling agents for some or all of the protein components that form a reaction system with a substance serving as an adsorbent for capturing the labeled protein components. A product prepared by the process of any one of claims 1