TWI464265B - Production method of a fusion protein in vivo - Google Patents

Description

Method for producing fusion protein in cells

The present invention relates to a method for producing a fusion protein, and in particular to a method for producing a fusion protein in a cell.

Fusion protein refers to a protein linked to at least two peptides, which may retain the original biological function of a single polypeptide or exhibit biological functions different from a single multi-peptide.

For example, a protein fused with β-galactosidase and galactose dehydrogenase has been shown to have an enzyme kinetic advantage over a single enzyme after extracellular experiments. (See Ljungcrantz. P., et al., Biochem., 1989, 28: 8786-8792). For example, a protein fused with trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase, after extracellular experiments, proves that it not only retains a single enzyme itself. In addition to its activity, it has an enzyme kinetic advantage over a single enzyme (see Seo HS, et al., Appl. Environ. Microbiol., 2000, 66: 2484-2490). For example, betaine aldehyde dehydrogenase-chloline dehydrogenase fusion protein can enhance the resistance of these organisms when expressed in Escherichia coli or Nicotiana tabacum . The ability to adversity (see Yilmaz JL, et al., Biotechnol. Progs., 2002, 18: 1176-1182).

Fusion proteins can naturally exist in cells, such as bcr-abl fusion proteins in cancer cells. Fusion proteins can also be purposefully present in cells by humans, genes Engineering has provided general experimental procedures to achieve such as polymerase chain reaction (PCR), enzyme digestion, DNA ligation, and the like. In detail, two or more gene fragments each encoding a multi-peptide are ligated together and fed into a cell. These multi-peptides are then expressed in the cells to form a fusion protein.

In terms of genetic engineering, although the method for preparing the fusion protein is quite simple, the fusion protein is mostly a macromolecule, so that its spatial elasticity and degree of freedom are hindered, and each other easily interferes with each other and cannot be folded, resulting in loss of the fusion protein. Biological activity, even in the body of the body to form an insoluble inclusion body. Therefore, the cell body is subsequently destroyed to obtain inclusion bodies, and the inactivated fusion protein is obtained from the inclusion body, and the inactivated fusion protein is folded and renatured in a suitable solution. However, the renaturation process is artificially operated after all, and is still no more than the environment provided by the cells in the body. Therefore, the biological function of the folded protein folded in the solution still has room for improvement.

In nature, anaerobic microorganisms evolve a rather special organelle that can effectively break down cellulose. Anaerobic microorganisms form a cellulosome outside the cell, the fibrous body contains a scaffolding protein, and the scaffold protein has a coherin domain, and the adhesion region binds to a cellulase. The dockerin domain, thereby allowing the fibrous body to become an enzyme complex and efficiently decompose cellulose (see Doi RH, et al., Nature Rev. Microbiol., 2004, 2: 541). -551).

The present invention is based on the fact that the adhesion region binds to the anchor region, and it is found that the two fusion proteins having the adhesion region and the anchor region are expressed in the same host cell, the adhesion region and the anchor. The fixed region does not easily affect the elasticity and degree of freedom of other polypeptides in the two fusion proteins, so that the fusion proteins can be folded and fused in the host cell, and provide excellent biological functions of the two fusion proteins.

Therefore, in a first aspect, the present invention discloses a method for producing a fusion protein comprising the steps of: (a) constructing a first nucleic acid molecule encoding an anchor region and a first target peptide; (b) constructing a a second nucleic acid molecule encoding an adhesion region and a second target peptide; (c) delivering the first nucleic acid molecule and the second nucleic acid molecule to a host cell; and (d) cultivating the host cell to express a first fusion a peptide and a second fusion peptide, and the first fusion peptide has an anchor region and a first target peptide, and the second fusion peptide has an adhesion region and a second target peptide, whereby the anchor region is attached to the adhesion a region to allow the first fusion peptide and the second fusion peptide to fuse with each other.

In a second aspect, the invention discloses a host cell comprising: a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes an anchor region and a first target peptide, the second nucleic acid molecule The code encodes an adhesion region and a second target peptide.

So far, the related research and application of the adhesion region and the anchoring region of the fibrous body have focused on the extracellular production of the fibrous body, and the obtained fibrous body is used for the decomposition of cellulose, but no research literature or patent literature has been found. The fusion proteins with the adhesion region and the anchor region are expressed in the same cell body, and the influence on the structure and biological function of the fusion proteins is not known.

After years of efforts and research by the present inventors, it has been found that the gene encoding the anchor region and the gene encoding the adhesion region are fed into the same cell body, and the two fusion proteins expressed by the cell body have interconnected anchor regions and adhesion regions, respectively. These fusion proteins are combined with each other, and these fusions Protein also does not form insoluble inclusion bodies. Moreover, such fusion proteins can be folded in the cell body, so that subsequent fusion of the fusion proteins in a solution can be used in different fields such as disease treatment and/or prevention, biomass materials and / or energy development, waste decomposition, environmental protection, ... and so on.

Therefore, the method for producing a fusion protein provided by the present invention comprises the steps of: (a) constructing a first nucleic acid molecule encoding an anchor region and a first target peptide; (b) constructing a code-adhesion region and a a second nucleic acid molecule of the second target peptide; (c) delivering the first nucleic acid molecule and the second nucleic acid molecule to a host cell; and (d) cultivating the host cell to express a first fusion peptide and a second a fusion peptide, wherein the first fusion peptide has an anchor region and a first target peptide, and the second fusion peptide has an adhesion region and a second target peptide, whereby the anchor region is attached to the adhesion region to allow the first The fusion peptide and the second fusion peptide are fused to each other.

The term "peptide" as used herein, alone or in combination, means a polymer composed of a plurality of amino acids and comprises a known naturally occurring amino acid or artificial chemical mimic. And the term "peptide" as used herein may be used interchangeably with terms such as "polypeptide" and "protein".

In particular, the term "target peptide" used herein, alone or in combination, means a selected peptide, and the selected peptide has known biological functions, such as compound catalysis, intracellular or extracellular signaling, antigen recognition. ,...and many more.

The term "nucleic acid" or "nucleic acid molecule" as used herein, alone or in combination, means a polymer consisting of nucleotides which may be single or double stranded and comprising known naturally occurring nucleotides or artificial imitations. . The term "nucleic acid" or "nucleic acid molecule" as used herein may be used interchangeably with terms such as "gene fragment", "gene", and "DNA".

Unless otherwise indicated, those skilled in the art will appreciate that the nucleic acid sequences set forth herein are not limited to the particular nucleotide sequences shown, but also to their complementary sequences. Furthermore, since the codons (consisting of three nucleotides) in the nucleic acid sequence are degenerate, and different codons can encode the same amino acid, those skilled in the art can understand the same peptide. A codon usage table obtains a degenerative sequence of a particular sequence. In other words, the nucleic acid sequences listed herein also encompass degenerate sequences of a particular sequence.

The term "host cell" as used herein, alone or in combination, encompasses subcultures or progeny of its subculture, in addition to the cells of a particular individual. However, all progeny may cause genetic modification during subsequent culture due to mutation or environmental factors, such that the progeny are inconsistent with the mother, but the progeny are still covered by the present invention. And the term "host cell" as used herein may be used interchangeably with terms such as "cell", "cell body", and "organism".

The anchoring region in step (a) of the present invention may be derived from, but not limited to, one of the following microorganisms: Acetivibrio cellulolyticus , Bacteroides cellulosolvens , Butyrivibrio fibrisolvens , Clostridium acetobutylicum , Clostridium cellobioparum , Clostridium cellulolyticum , Clostridium cellulovorans , Clostridium josui , Clostridium papyrosolvens , Clostridium thermocellum , Ruminococcus albus , Ruminococcus flavefaciens , Neocallimastix patriciarum , Orpinomyces joyonii , Orpinomyces PC-2 , Piomyces equi , Piomyces E2 .

The adhesion region in step (b) of the present invention may be derived from, but not limited to, one of the following microorganisms: Acetivibrio cellulolyticus , Bacteroides cellulosolvens , Butyrivibrio fibrisolvens , Clostridium acetobutylicum , Clostridium cellobioparum , Clostridium cellulolyticum , Clostridium cellulovorans , Clostridium josui , Clostridium papyrosolvens , Clostridium thermocellum , Ruminococcus albus , Ruminococcus flavefaciens , Neocallimastix patriciarum , Orpinomyces joyonii , Orpinomyces PC-2 , Piomyces equi , Piomyces E2 .

The construction process of the steps (a) and (b) of the present invention has been reported in detail in related research literature and patent documents in the technical field, such as Sambrook J., et al., 2001, Molecular Cloning: a Laboratory Manual, 3rd ed. Unless otherwise indicated, a detailed description of these two steps can be found in the aforementioned documents.

When the steps (a) and (b) of the present invention are carried out, the order of the two steps is not limited. That is, step (a) is performed first, followed by step (b); step (b) is performed first, then step (a) is performed; or both steps (a) and (b) are allowed at the same time.

According to a preferred embodiment of the present invention, when the step (a) of the present invention is carried out, the first nucleic acid molecule can be implanted into a vector which can be expressed by the host cell, and when the step (b) of the present invention is carried out, The second nucleic acid molecule is implanted into a vector implanted with the first nucleic acid molecule.

To control transcription of the first nucleic acid molecule and the second nucleic acid molecule, the vector further has a promoter operably linked to the first nucleic acid molecule and the second nucleic acid molecule. The phrase "operably linked" as used herein means that the two gene segments are quite close and one of the fragments is sufficient to affect the other. In the preferred embodiment, the promoter is located at the 5' end of the first nucleic acid molecule and the second nucleic acid molecule.

In this preferred embodiment, the promoter may be a constitutive promoter or an inducible promoter. The promoter is determined in accordance with the host cell, and may be derived from one of a virus, a bacterial cell, a yeast cell, a fungal cell, an algal cell, a plant cell, an animal cell, an insect cell, or a human cell. Promoters derived from bacterial cells, such as the tac promoter, the T7 promoter, the T7 A1 promoter, the lac promoter, the trp promoter, the trc promoter, the ara BAD promoter or the lambda P _R P _L promoter. A promoter derived from a plant cell, such as a 35S CaMV promoter, an actin promoter, or a ubiquitin promoter. A promoter derived from an animal cell, such as the SV40 promoter, the RSV promoter, the CMV promoter or the HSV promoter.

The vector further contains other expression control elements to further control transcription of the first nucleic acid molecule and the second nucleic acid molecule, such as a transcription start site, a transcription stop site, and a ribosome binding site ( A ribosome binding site), an RNA splicing site, a polyadenylation site, and a translation stop site. These performance control factors are determined by the host cell.

The vector also contains a marker gene or reporter gene for screening the vector. The marker gene is determined according to the host cell, such as the dihydrofolate reductase gene of the eukaryotic cell, the anti-G418 gene or the neomycin gene, and the ampicillin of the bacterial cell. Gene, anti-streptomycin gene or anti-kanamycin gene. The gene is reported, such as a green fluorescent protein gene, a luciferase gene, a β-galactosidase gene or a chloramphenicol acetyltransferase gene.

According to another preferred embodiment of the present invention, when performing step (a) of the present invention, the first nucleic acid molecule can be implanted into a first carrier which can be expressed by the host cell, and when step (b) of the present invention is carried out, The second nucleic acid molecule can be implanted into a second vector that can be expressed by the host cell.

To control transcription of the first nucleic acid molecule and the second nucleic acid molecule, the first vector further has a first promoter operably linked to the first nucleic acid molecule, and the second vector further has an operably linked second nucleic acid The second promoter of the molecule. In the preferred embodiment, the first promoter is located at the 5' end of the first nucleic acid molecule and the second promoter is located at the 5' end of the second nucleic acid molecule.

In the preferred embodiment, the first promoter and the second promoter may each be a combinatorial promoter or an inducible promoter. The first promoter and the second promoter are determined according to the host cell, and may be derived from one of a virus, a bacterial cell, a yeast cell, a fungal cell, an algal cell, a plant cell, an animal cell, an insect cell, and a human cell. By. A promoter derived from a bacterial cell, such as a tac promoter, a T7 promoter, a T7 A1 promoter, a lac promoter, a trp promoter, a trc promoter, an ara BAD promoter, or a lambda P _R P _L promoter. A promoter derived from a plant cell, such as the 35S CaMV promoter, an actin promoter or a ubiquitin promoter. A promoter derived from an animal cell, such as the SV40 promoter, the RSV promoter, the CMV promoter or the HSV promoter.

The first vector and the second vector each further contain other expression control elements to further control transcription of the first nucleic acid molecule and the second nucleic acid molecule, such as a transcription start position, a transcription termination position, a ribosome binding position, and an RNA cutter. The cleavage position, a polyadenylation position, and a translation termination position. These performance control factors are determined by the host cell.

The first vector and the second vector each additionally contain a marker gene or reporter gene for screening these vectors. The marker gene is determined according to the host cell, such as the dihydrofolate reductase gene of the eukaryotic cell, the anti-G418 gene or the anti-neomycin gene, the anti-ampicillin gene of the bacterial cell, the anti-streptomycin gene or the anti-Kang Natamycin gene. Reporting genes, such as the green fluorescent protein gene, the luciferase gene, the β-galactosidase gene, or the chloramphenicol acetyltransferase gene.

The host cell in step (c) of the present invention may be a prokaryotic cell or a eukaryotic cell. Examples of prokaryotic cells may be, but are not limited to, bacteria, blue-green algae or radiobacteria, and representative examples of the bacteria are Escherichia coli, Bacillus subtilis , Lactobacillus sp. , Streptomyces. Sp. ) or Salmonella typhimurium .

Examples of eukaryotic cells can be, but are not limited to, fungal cells, protozoan cells, plant cells, animal cells, insect cells, or human cells. Representative examples of fungal cells are yeast cells such as baker's yeast or methanolophilus yeast. Plant cells can be obtained from gymnosperms or angiosperms. Further, the plant cell can be isolated from the root, stem, leaf or meristematic tissue of the above plant and cultured in the form of a protoplast or a healing tissue. Representative examples of insect cells are Drosophila S2 cells or S. cerevisiae Sf21 cells or Sf9 cells. The animal cells are not limited to cultured cells or cells in vivo, preferably from vertebrate animals, and more preferably from mammals. Moreover, the animal cells can be isolated from organs or tissues such as kidney, liver, lung, ovary, breast, skin, bone, muscle or skin, and examples thereof can be CHO, COS, BHK, HEK-293, Hela, NIH3T3, VERO, MDCK, MOLT-4, Jurkat, K562 or HepG2.

Generally, when the host cell is a prokaryotic cell, the delivery process of step (c) of the present invention is referred to as "transformation." Those skilled in the art should understand how to implement transformation, such as using calcium carbonate, calcium chloride as a medium, gene gun, phage infection, and therefore will not be described again.

Generally, when the host cell is a eukaryotic cell, the delivery process of step (c) of the present invention is referred to as "transfection." Those skilled in the art will understand how to perform transfection, such as the use of liposome as a medium, electroporation, microiniection, animal and plant viral infections, and therefore will not be described again.

The culture process of step (d) of the present invention is a host cell selected by those skilled in the art. This can be easily accomplished using suitable media and culture conditions. For example, when the host cell is Escherichia coli, the host cell is cultured in LB medium and placed in a 30 ° C incubator in a shaking manner.

Also included within the scope of the present invention is a host cell comprising: a first nucleic acid molecule encoding an anchor region and a first target peptide; and a first encoding an adhesion region and a second target peptide Two nucleic acid molecules.

It is specifically noted that the host cells, anchoring regions, and adhesion regions mentioned herein are the same as those mentioned in the foregoing manufacturing methods, and therefore will not be described again.

The invention is further illustrated by the following examples.

<Experimental methods and materials>

The methods employed in the following examples are primarily based on textbooks familiar to those skilled in the art: Sambrook J., et al., 2001, Molecular Cloning: a Laboratory Manual, 3rd ed.

The bacterial culture solution concentration was measured using a spectrophotometer (V530, Jasco) using a wavelength of 550 nm, and the measured absorbance was recorded as OD ₅₅₀ . The total protein concentration was measured using a protein assay reagent (BioRad Co.). The target protein concentration was determined by analyzing the electrophoretically separated protein using an image analyzer (AlphaImager EP, Alpha Innotech Co.).

The purification of bacterial chromosomes, plasmids and DNA was performed using Wizard Genomic DNA Purification Kit (Promega Co.), High-Speed Plasmid Mini Kit (Geneaid Co.) and Gel/PCR DNA Fragments Extraction Kit (Geneaid, respectively). Co.) and other commercial groups. Restriction enzymes were purchased from New England Biolabs and Fermentas Life Science. T4 ligase and Pfu DNA polymerase were purchased from Promega Co. The primer used for the polymerase chain reaction was commissioned by Mingxin Biotechnology Co., Ltd. (Taipei) and Yuanzi Biotech Co., Ltd. North) synthetic.

DNA transfer was performed by chemical transformation, and the detailed procedure was as follows: Single colonies were selected from LB solid medium to LB liquid medium, and cultured at a suitable temperature for 12 to 16 hours at 150 rpm. The bacterial solution was inoculated into fresh LB liquid medium at an initial concentration of OD ₅₅₀ = 0.08, and cultured at a suitable temperature at 150 rpm. Until the bacterial concentration OD ₅₅₀ = 0.3-0.5, 4 mL of the bacterial solution was taken out into a sterile test tube, ice-bathed for 10 minutes, then centrifuged at 4,000 rpm for 2 minutes and the supernatant was removed. The cells remaining after centrifugation were uniformly mixed with 2 mL of 0.1 M MgCl ₂ , and then ice-bathed for 5 minutes, centrifuged at 4,000 rpm for 2 minutes, and the supernatant was removed. The cells remaining after centrifugation were uniformly mixed with 1.5 mL of 0.05 M CaCl ₂ , and then ice-bathed for 20 minutes, centrifuged at 4,000 rpm for 2 minutes, and the supernatant was removed. After 300 μL of 0.05 M CaCl _{2 was added} and uniformly mixed with the centrifuged cells, a competent cell was prepared. Next, 2 ng/mL of plastids and 100 μL of competent cells were added to a sterile test tube and mixed well. After 30 minutes in an ice bath, the sterile test tube was transferred to a 42 ° C constant temperature water bath for 2 minutes and then ice bathed for 5 minutes. Finally, 1 mL of fresh LB liquid medium (4 ° C) was added to the competent cell bacteria solution, and the cells were incubated at an appropriate temperature for 2 hours, centrifuged at 4,000 rpm for 10 minutes, and the supernatant was removed. After mixing the remaining LB liquid medium with the centrifuged competent cell bacteria, take appropriate amount of competent cell bacteria solution, uniformly apply it to the LB solid medium containing antibiotics, and place it in a constant temperature incubator at an appropriate temperature. Cultivate overnight to grow colonies.

The enzyme activity was measured by high-pressure liquid chromatography (HPLC). The activity test of Agrobacterium radiobacter B11291 D-carbamoylase (AHL) is to mix the sample with the reaction solution (final concentration: 20 mM CpHPG and 10 mM sodium phosphate buffer, pH 6.6-6.8). The reaction was carried out at 40 ° C for 30 minutes. The reaction mixture was heated at 100 ° C for 10 minutes to terminate the reaction, and then centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken for HPLC analysis. The column used for HPLC analysis was a C18 column (250 mm × 4.6 mm, Bos Hypersil), and the mobile phase used was a solution of 2 mM ammonium acetate (pH 3.2): methanol = 91:9 by volume, and was carried out at a wavelength of 280 nm UV. Signal detection. The activity test of Agrobacterium radiobacter B11291 D-hydantoinase (HDT) is similar to that of AHL except that the reaction solution used is a final concentration: 10 mM HPH, 0.1 M Tris buffer, pH 8.0 and 0.5 mM MnCl ₂ solution. . Enzyme activity is defined as enzyme activity per unit (U) = product production (μmole) / reaction time (min).

<Example 1: Construction of plastid pETW-A2HCh>

As shown in Fig. 1, the plastid pETW-A2HCh contains a T7 promoter, a gene fragment driven by a promoter to encode Agrobacterium radiobacter B11291 AHL and HDT (AHL-HDT) fusion protein, and a ColE1 origin of replication of Escherichia coli (replication origin) ), anti-ampicillin gene. The construction process is as follows. First, according to the nucleic acid sequence of Agrobacterium radiobacter B11291 AHL (registration number: U59376) in the National Center for Biotechnology Information (NCBI) genomic database, the primer is introduced: Forward introduction: 5' -tttgt gatatc gctaccaccaccaccggtcaggccaagccaagcttg-3' (SEQ ID NO: 1); reverse primer: 5'-taact tctaga aggagatatacatatgac-3' (SEQ ID NO: 2).

The forward primer is designed with the shear position of the restriction enzyme EcoR V, and the reverse primer is designed with the shear position of Xba I (as indicated by the bottom line). Using plastid pChA203 (see Chiang CJ et al., J. Agri. Food Chem., 2008, 56: 6348-6354) as a template, and using this two primers for PCR reaction, a gene fragment encoding AHL was amplified. 0.96kb). After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the gene fragment obtained by cleavage of the restriction enzymes EcoR V and Xba I was used, and the fragmented gene fragment was recovered using the Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plastid pHDT was purified using a High-Speed Plasmid Mini kit (see Chem JT et al., J. Biotechnol., 2005, 117: 267-275). After the plastid pHDT was sheared using the restriction enzymes EcoR V and Xba I, the sheared plastid pHDT was recovered using a Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product is sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pETW-A2HCh, which encodes AHL-HDT. A gene fragment of the fusion protein is shown in SEQ ID NO: 3.

<Example 2: Construction of plastid pTH-ChA203>

As shown in Figure 2, plastid pTH-ChA203 contains a T7 promoter, which is driven by a promoter to encode Agrobacterium radiobacter B11291 AHL and Bacillus circulans WL-12 chitin binding domain (ChBD) (AHL-ChBD) fusion. A gene fragment of the protein, a pSC101 origin of replication of E. coli, and an anti-connamycin gene. The construction process is as follows. First, the primer is designed according to the nucleic acid sequence of pTH18Kr in the genomic database of the National Center for Biotechnology Information (registration number: AB019603): forward primer: 5'-taacc agatct gattagaaaaactcatcg-3' (SEQ ID NO: 5); reverse primer: 5'-agaac ctgcag tcagatccttccgtatttagc-3' (SEQ ID NO: 6).

The forward primer is designed with the restriction position of the restriction enzyme Bgl II, and the reverse primer is designed with the shear position of Pst I (as indicated by the bottom line). The plastid pTH18Kr was used as a template, and the PCR reaction was carried out using the two primers, and a gene fragment (about 2.5 kb) having a pSC101 origin of replication and an anti-connamycin gene was amplified. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the gene fragment obtained by cleavage of the restriction enzymes Bgl II and Pst I was used, and the fragmented gene fragment was recovered using the Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plasmid pChA203 was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pChA203 using the restriction enzymes Bgl II and Pst I, the spliced plastid pChA203 was recovered using a Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product is sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pTH-ChA203, which encodes AHL-ChBD. A gene fragment of the fusion protein is shown in SEQ ID NO: 7.

<Example 3: Construction of plastid pTH-AL203Coh>

As shown in Figure 3, the plastid pTH-AL203Coh contains a T7 promoter, a gene fragment driven by a promoter to encode the Agrobacterium radiobacter B11291 AHL and Clostridium thermocellum adhesion region CohI (AHL-CohI) fusion protein, and the pSC101 replication initiation of Escherichia coli Point, anti-connamycin gene. The construction process is as follows. First, according to the nucleic acid sequence of Clostridium thermocellum cipA gene in the genomic database of the National Center for Biotechnology Information (registration number: X67406), the primer is introduced: forward primer: 5'-atcat ctcgag ttatgcggccgcaagctttggtg-3' (SEQ ID NO: 9); reverse primer: 5'-atta gaattc agatctcagccaaatgttcc-3' (SEQ ID NO: 10).

The forward primer is designed with the shear position of the restriction enzyme Xho I, and the reverse primer is designed with the shear position of EcoR I (as indicated by the bottom line). The chromosome of the plastid Clostridium thermocellum DSM1237 (taken from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) was used as a template, and the PCR reaction was carried out using the two primers to increase a gene fragment (about 0.5 kb) encoding the adhesion region CohI. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the amplified gene fragment was amplified by restriction enzymes Xho I and EcoR I, and the fragmented gene fragment was recovered using a Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plastid pTH-ChA203 was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pTH-ChA203 using the restriction enzymes Xho I and EcoR I, the spliced plastid pTH-ChA203 was recovered using a Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pTH-AL203Coh, which encodes AHL-CohI. A gene fragment of the fusion protein is shown in SEQ ID NO:11.

<Example 4: Construction of plastid pTac-HDTDoCh>

As shown in Figure 4, the plastid pTac-HDTDoCh contains a Tac promoter, which is driven by a promoter to encode a thioredoxin (TrxA), an Agrobacterium radiobacter B11291 HDT, a Clostridium thermocellum anchor region Doc1, and a Bacillus circulans WL-12 ChBD ( TrxA-HDT-Doc1-ChBD) gene fragment of fusion protein, ColE1 origin of replication of Escherichia coli, and ampicillin resistance gene. The construction process is as follows. First, the primer is designed according to the nucleic acid sequence of plastid pET32a (Novagen Co.): forward primer: 5'-ggtgaataattttatcgctcat c tgtatatctccttctagaggg-3' (SEQ ID NO: 13); reverse primer: 5'- Ccctctagaaggagatatacagatgagcgataaaattattcacc-3' (SEQ ID NO: 14).

Both primers were designed with the cleavage position of the mutant restriction enzyme Nde I (as indicated by the bottom line). The plastid pET32a was used as a template, and the PCR reaction was carried out using the QuickChange Site-Directed Mutagenesis Kit (Strategene Co.) and the two primers to increase the plastid pET32a with the first Nde I cleavage position mutation downstream of the T7 promoter. Gene fragment. After the amplified gene fragment was purified by Gel/PCR DNA Fragments Extraction Kit, the purified DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain a plastid pET32-N. Next, after cleavage of the plastid pET32-N using the restriction enzymes Xho I and Nde I, the spliced plastid pET32-N was recovered using a Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plastid pChHDT was purified using a High-Speed Plasmid Mini kit (see Chem JT et al., J. Biotechnol., 2005, 117: 267-275). After cleavage of the plastid pChHDT using the restriction enzymes Xho I and Nde I, the spliced plastid pChHDT was recovered using the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments with T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pET-TrHDTCh.

Secondly, according to the nucleic acid sequence of Clostridium thermocellum cipA gene in the genomic database of the National Center for Biotechnology Information (registration number: X67406), the primer was introduced: forward primer: 5'-tgtga ctcgag ttagagctcgttcttgtacggcaatgtatc-3' (SEQ ID NO: 15) Reverse primer: 5'-gcatg aagctt aaagtacctggtactccttc-3' (SEQ ID NO: 16).

The forward primer is designed with the shear position of the restriction enzyme Xho I, and the reverse primer is designed with the shear position of Hind III (as indicated by the bottom line). Using the chromosome Clostridium thermocellum DSM1237 as a template, and using the two primers for PCR reaction, a gene fragment encoding the anchor region Doc1 (about 0.23 kb) was amplified. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the gene fragment obtained by restriction amplification with restriction enzymes Xho I and Hind III was used, and the fragmented gene fragment was recovered using the Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plasmid pET-TrHDTCh was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pET-TrHDTCh using the restriction enzymes Xho I and Hind III, the spliced plastid pET-TrHDTCh was recovered using the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments with T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pET-TrHDTDo.

Then, the primer was designed according to the nucleic acid sequence of the plastid pET-TrHDTCh: forward primer: 5'-agatgcttttctgtgactgg-3' (SEQ ID NO: 17); reverse primer: 5'-agcat gagctc ggcctgaccggtctgaac-3' (SEQ ID NO: 18).

The reverse primer is designed with the Sic I shear position (as indicated by the bottom line). Using the plastid pET-TrHDTCh as a template, and using the two primers for PCR reaction, a gene fragment encoding ChBD (about 1.3 kb) was amplified. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the amplified gene fragment was cleaved using restriction enzyme Sac I, and the fragmented gene fragment was recovered using a Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plasmid pET-TrHDTDo was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pET-TrHDTDo using the restriction enzyme Sac I, the spliced plastid pET-TrHDTDo was recovered using the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments with T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pET-HDTDoCh.

Next, the primer was designed according to the nucleic acid sequence of the plastid pJF-Trxfus (see Chao YP, Appl. Microbiol. Biotechnol., 2000, 54: 348-353): forward primer: 5'-gaatt tctaga cctgtgtgaaattgttatcc-3' ( SEQ ID NO: 19); reverse primer: 5'-gagtgagatatttatgccag-3' (SEQ ID NO: 20).

The forward guide is designed with the Xba I cut position (as indicated by the bottom line). The plastid pJF-Trxfus was used as a template, and the PCR reaction was carried out using the two primers to increase a gene fragment (about 1 kb) having a Tac promoter. After the amplified gene fragment was purified using the Gel/PCR DNA Fragments Extraction Kit, the gene fragment obtained by cleavage of the restriction enzymes Xba I and Apa I was used, and the fragmented gene fragment was recovered using the Gel/PCR DNA Fragments Extraction Kit. On the other hand, the plasmid pET32-N was purified using a High-Speed Plasmid Mini kit. After cleavage of the plastid pET32-N using the restriction enzymes Xba I and Apa I, the spliced plastid pET32-N was recovered using the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pTac-N.

Finally, after cleavage of the plastid pTac-N using the restriction enzymes Xba I and Pst I, the spliced plastid pTac-N was recovered using the Gel/PCR DNA Fragments Extraction Kit. In another part, the restriction enzyme Xba I and Pst I were used to cleave the plastid pET-HDTDoCh to remove the T7 promoter of the plastid pET-HDTDoCh, and the remaining gene fragments were recovered by the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain a plastid pTac-HDTDoCh encoding TrxA-HDT. A gene fragment of the -Doc1-ChBD fusion protein is shown in SEQ ID NO:21.

<Example 5: Construction of plastid pTac-TrChHDT>

As shown in Fig. 5, the plastid pTac-TrChHDT contains a Tac promoter, a gene fragment driven by a promoter to encode a TrxA, Agrobacterium radiobacter B11291 HDT, Bacillus circulans WL-12 ChBD (TrxA-HDT-ChBD) fusion protein, and Escherichia coli. The ColE1 replication initiation point, the ampicillin resistance gene. The construction procedure was as follows. The restriction enzymes Xba I and Pst I were used to cleave the plastid pTac-HDTDoCh to retain the Tac promoter of the plastid pTac-HDTDoCh, and the remaining gene fragments were recovered by the Gel/PCR DNA Fragments Extraction Kit. On the other hand, after the plastid pET-TrHDTCh was cleaved with the restriction enzymes Xba I and Pst I to remove the T7 promoter of the plastid pET-TrHDTCh, the remaining gene fragments were recovered by the Gel/PCR DNA Fragments Extraction Kit. After binding the above-mentioned two recovered gene fragments by T4 ligase, the ligated DNA product was sent to Escherichia coli DH5α by the above-mentioned "chemical transformation method" to obtain plastid pTac-TrChHDT, which encodes TrxA-HDT. A gene fragment of the -ChBD fusion protein is shown in SEQ ID NO:23.

<Example 6: Expression of AHL-HDT fusion protein>

The plastid pETW-A2HCh was sent to Escherichia coli BL21 (DE3) by the above "chemical transformation method" to obtain a recombinant strain BL21(DE3)/pETW-A2HCh. A single colony of the recombinant strain was selected from LB solid medium to LB medium (5 mL) containing 50 μg/mL ampicillin, and cultured in a constant temperature shaking box at 30 ° C and 150 rpm for 16 hours, and then inoculated to freshly containing 50 μg / mL of amine. LB broth (25 mL) of benzylpenicillin was brought to a starting concentration of OD ₅₅₀ = 0.08. Next, the recombinant strain was continuously cultured in a constant temperature shaking box at 30 ° C and 150 rpm until its concentration reached OD ₅₅₀ = 0.3, and 50 μM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to induce recombinant strain to produce protein. After continuing to culture the recombinant strain for 4 hours, the recombinant strain was collected and the bacteria were washed with 0.01 M sodium phosphate buffer (pH 7.5). Finally, the bacteria were suspended in 1 mL of sodium phosphate buffer so that the bacterial concentration reached OD ₅₅₀ = 10, and the bacteria were broken by ultrasonic waves, and the produced protein was analyzed by electrophoresis using non-original sodium dodecyl sulfate-polyacrylamide. . As shown in Fig. 6, after IPTG induction, the recombinant strain BL21(DE3)/pETW-A2HCh produced a large amount of the AHL-HDT fusion protein, but it formed an insoluble inclusion body (diameter 5 of Fig. 6).

<Example 7: Expression of AHL-ChBD fusion protein, TrxA-HDT-ChBD fusion protein>

The plastids pTH-ChA203 and pTac-TrChHDT were simultaneously sent to E. coli BAD-5 by the above-mentioned "chemical transformation method" (see Mang ZW et al., J. Agri. Food Chem., 2011, 59:6534-6542). The recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT was obtained. A single colony of the recombinant strain was selected from LB solid medium to LB medium (5 mL) containing 50 μg/mL ampicillin and 50 μg/mL connamycin, and then cultured in a constant temperature shaking box at 30 ° C and 150 rpm for 16 hours. LB medium (25 mL) freshly containing 50 μg/mL ampicillin and 50 μg/mL connamycin was inoculated to a starting concentration of OD ₅₅₀ = 0.08. Next, the recombinant strain was continuously cultured in a constant temperature shaking box at 30 ° C and 150 rpm until its concentration reached OD ₅₅₀ = 0.3, and 30 μM L-arabinose was added to induce recombinant strain to produce protein. After continuing to culture the recombinant strain for 4 hours, the recombinant strain was collected and the bacteria were washed with 0.01 M sodium phosphate buffer (pH 7.5). Finally, the bacteria were suspended in 1 mL of sodium phosphate buffer so that the bacterial concentration reached OD ₅₅₀ = 10, and the bacteria were broken by ultrasonic waves, and the produced protein was analyzed by electrophoresis using non-original sodium dodecyl sulfate-polyacrylamide. . As shown in Fig. 7, after arabinose induction, the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT can produce a large amount of soluble AHL-ChBD fusion protein and TrxA-HDT-ChBD fusion protein (diameter 2 of Fig. 7). .

<Example 8: Expression of AHL-CohI fusion protein, TrxA-HDT-Doc1-ChBD fusion protein>

The plastids pTH-AL203Coh and pTac-HDTDoCh were simultaneously sent to E. coli BAD-5 by the above-mentioned "chemical transformation method" to obtain a recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh. A single colony of the recombinant strain was selected from LB solid medium to LB medium (5 mL) containing 50 μg/mL ampicillin and 50 μg/mL connamycin, and then cultured in a constant temperature shaking box at 30 ° C and 150 rpm for 16 hours. LB medium (25 mL) freshly containing 50 μg/mL ampicillin and 50 μg/mL connamycin was inoculated to a starting concentration of OD ₅₅₀ = 0.08. Next, the recombinant strain was continuously cultured in a constant temperature shaking box at 30 ° C and 150 rpm until its concentration reached OD ₅₅₀ = 0.3, and 30 μM L-arabinose was added to induce recombinant strain to produce protein. After continuing to culture the recombinant strain for 4 hours, the recombinant strain was collected and the bacteria were washed with 0.01 M sodium phosphate buffer (pH 7.5). Finally, the bacteria were suspended in 1 mL of sodium phosphate buffer so that the bacterial concentration reached OD ₅₅₀ = 10, and the bacteria were broken by ultrasonic waves, and the produced protein was analyzed by electrophoresis using non-original sodium dodecyl sulfate-polyacrylamide. . As shown in Fig. 7, after arabinose induction, the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh can produce a large amount of soluble AHL-ChoI fusion protein and TrxA-HDT-Doc1-ChBD fusion protein (Fig. 7). 3).

<Example 9: Chitin adsorption analysis of protein>

The plastids pTH-AL203Coh and pTac-TrChHDT were simultaneously sent to E. coli BAD-5 by the above-mentioned "chemical transformation method" to obtain a recombinant strain BAD-5/pTH-AL203Coh/pTac-TrChHDT. The recombinant strain was cultured to produce a protein according to the method of Example 7 or 8.

In the past, the combination of ChBD and chitin in Bacillus circulans WL-12 chitinase A1 was very specific and stable, and could be combined in a wide pH range (see Hashimoto et al., J. Bacteriol., 2000, 182: 3045-3054; and Chern JT et al., J. Biotechnol., 2005, 117: 267-275). In this example, the fusion protein was adsorbed by chitin beads, and the operation procedure was as follows: the bacteria were centrifuged, and the bacteria were washed twice with 0.01 M sodium phosphate buffer (pH 7.5). The bacteria were suspended in 1 mL of sodium phosphate buffer so that the bacterial concentration reached OD ₅₅₀ = 10, and the bacteria were broken by ultrasonic waves, centrifuged, and the supernatant was recovered to obtain a cell extract. For the other part, 0.5 g of chitin spheres (New England Biolabs) were taken into a 1.5 mL centrifuge tube. After washing the chitin balls in 1 mL of 0.01 M sodium phosphate buffer (pH 7.5), the chitin balls were allowed to settle at the bottom of the centrifuge tube and the supernatant was removed, and the above procedure was repeated three times. Next, the cell extract containing 50 μg of the protein was added to the washed chitin spheres, and the adsorption reaction was carried out at 4 °C. After reacting for 12 hours, wait for the chitin ball to settle at the bottom of the centrifuge tube, then remove the supernatant and wash it twice with 1 mL of 0.01 M sodium phosphate buffer (pH 7.5) to wash away without adsorption. The protein of chitin balls. Adding a 2% SDS solution to the protein adsorbed to the chitin spheres and heating at 100 ° C for 5 to 10 minutes to separate the adsorbed protein from the chitin spheres, using the non-organic sodium dodecyl sulfate-polypropylene The adsorbed protein was analyzed by guanamine electrophoresis.

As shown in Fig. 8(A), after the adsorption reaction, the amount of TrxA-HDT-ChBD fusion protein produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-TrChHDT was significantly reduced in the cell extract (path 2). 3). It is said that the TrxA-HDT-ChBD fusion protein can be adsorbed by chitin spheres due to ChBD. Conversely, the amount of AHL-CohI fusion protein produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-TrChHDT did not change significantly in the cell extract before and after the adsorption reaction (paths 2 and 3). It is said that the AHL-CohI fusion protein cannot be adsorbed by chitin balls because it has no ChBD. In addition, after heating to separate the adsorbed protein from the chitin spheres, only the TrxA-HDT-ChBD fusion protein (path 6) was obtained, indicating that there was no effect between the TrxA-HDT-ChBD fusion protein and the AHL-CohI fusion protein. Therefore, only the TrxA-HDT-ChBD fusion protein can be adsorbed by chitin spheres.

As shown in Fig. 8(B), after the adsorption reaction, the AHL-CohI fusion protein produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh, The amount of TrxA-HDT-Doc1-ChBD fusion protein residues in the cell extract was significantly reduced (both 2, 3). This means that the AHL-CohI fusion protein and the TrxA-HDT-Doc1-ChBD fusion protein can be adsorbed by chitin spheres. However, it is worth noting that the AHL-CohI fusion protein does not contain ChBD but is adsorbed by chitin spheres. After heating to separate the adsorbed protein from the chitin sphere, the AHL-CohI fusion protein and the TrxA-HDT-Doc1-ChBD fusion protein (path 6) were obtained, indicating that the adhesion region CohI and TrxA-HDT contained in the AHL-CohI fusion protein were expressed. - The adhesion between the anchor region Doc1 contained in the -Doc1-ChBD fusion protein is such that the ChBD contained in the TrxA-HDT-Doc1-ChBD fusion protein is adsorbed by the chitin sphere, and the AHL-CohI fusion protein is indirectly Adsorbed by chitin balls.

<Example 10: Activity analysis of protein>

After the culture of the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT of Example 7 and the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh of Example 8 was completed, the cells were collected by centrifugation at 4 °C. When the bacterial concentration was adjusted to OD ₅₅₀ = 10 using deionized water, the bacteria were disrupted by ultrasonic waves, centrifuged at 4 ° C and 12,000 rpm, and the supernatant was taken to obtain a cell extract.

The enzyme activities of AHL and HDT were measured by the above-mentioned "enzyme activity" test. First, a cell extract containing 5 μg of protein was added to 1 mL of the reaction solution, and after the reaction, the concentration of the reactant in the solution was analyzed using a high-performance liquid chromatograph. In another part, individual proteins in the cell extracts were analyzed by electrophoresis using non-original sodium dodecyl sulfate-polyacrylamide, and the AHL and HDT enzyme activities of individual proteins were determined by an image analyzer. In a further portion, a cell extract containing 15 μg of protein was taken into a 1 mL reaction solution containing 20 mM DL-hydroxylphenyl hydantoin (DL-HPH) and 0.5 mM sodium phosphate buffer (pH 7.5), and Enzymatic tandem connection of AHL and HDT in cell extract at 40 °C The reaction produces D-p-hydroxyphenylglycine (D-HPG). The D-HPG concentration (mM) generated in the solution was analyzed using a high-performance liquid chromatograph, and the concentration was divided by the reaction time to obtain a total reaction rate.

As shown in Table 1, the AHL-ChBD fusion protein and the TrxA-HDT-ChBD fusion protein produced in Example 7 each exhibited an AHL-CohI fusion protein, TrxA-HDT-Doc1-ChBD fusion protein which was superior to that produced in Example 8. HDT activity and AHL activity. However, the AHL-CohI fusion protein produced in Example 8 and the TrxA-HDT-Doc1-ChBD fusion protein had a total reaction rate 1.6 times higher than that of the AHL-ChBD fusion protein produced in Example 7, and the TrxA-HDT-ChBD fusion protein. This result indicates that the AHL-CohI fusion protein produced in Example 8 and the TrxA-HDT-Doc1-ChBD fusion protein are combined with each other to form an enzyme complex through the adhesion region CohI and the anchor region Doc1, resulting in a so-called " The metabolite channeling phenomenon effectively increases the reaction rate.

<Example 11: Biotransformation analysis of strain>

In this embodiment, the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT of Example 7 and the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh of Example 8 were used for biotransformation reaction, mainly catalyzing D- HPG generation.

After the recombinant strain of Example 7 and the recombinant strain of Example 8 were cultured, the cells were collected by centrifugation at 4 ° C, and the cells were washed twice with 0.01 M sodium phosphate buffer (pH 7.5). After the cells were suspended in 1 mL of sodium phosphate buffer (pH 7.5), the cells were added to 10 mL of a reaction solution containing 100 mM DL-HPH and 0.5 mM sodium phosphate buffer (pH 7.5) to finally The bacterial concentration was OD ₅₅₀ = 30, and a conversion reaction was carried out at 40 ° C for 10 hours to form D-HPG. After completion of the reaction, the reaction solution was centrifuged at 12,000 at normal temperature, and the supernatant liquid was taken out for HPLC analysis. After washing the centrifuged cells twice with 0.01 M sodium phosphate buffer (pH 7.5), the washed cells were again centrifuged and added to 10 mL of the reaction solution to carry out a new transformation reaction, and several times. Repeat the above steps.

As shown in Fig. 9(A), the recombinant strain of Example 7 can be used only once to achieve 100% D-HPG conversion; and when it is repeatedly used for the second time, D-HPG conversion is lowered. Up to 80%. As shown in Fig. 9(B), the recombinant strain of Example 8 can be used repeatedly for 4 times to achieve 100% D-HPG conversion; and when it is repeatedly used for the 5th time, the D-HPG conversion rate is still Maintain at around 80%. This result indicates that the AHL-CohI fusion protein produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh, the TrxA-HDT-Doc1-ChBD fusion protein will pass through the adhesion region CohI and the anchor region Doc1, but within the cell. Combining with each other to form an enzyme complex, compared to individual proteins that do not bind to each other (such as the AHL-ChBD fusion protein produced by the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT, TrxA-HDT-ChBD fusion protein), The enzyme complex apparently has a higher stability and enhances the efficiency of the biotransformation reaction.

Obviously, many modifications and differences may be made to the invention in light of the above description. It is therefore to be understood that within the scope of the appended claims, the invention may be The above is only preferred of the present invention. The examples are not intended to limit the scope of the invention, and all equivalent modifications or modifications may be made without departing from the spirit of the invention.

Figure 1 is a map of plastid pETW-A2HCh, abbreviated symbol: Ap, anti-ampicillin gene; T7 promoter, T7 promoter; AHL, gene encoding Agrobacterium radiobacter B11291 AHL; HDT, gene encoding Agrobacterium radiobacter B11291 HDT ; ColE1, the starting point of ColE1 replication in E. coli.

Figure 2 is a map of plastid pTH-ChA203, abbreviated symbol: Km, anti-connamycin gene; T7 promoter, T7 promoter; AHL, gene encoding Agrobacterium radiobacter B11291 AHL; ChBD, encoding Agrobacterium radiobacter WL-12 The gene for ChBD; pSC101, the origin of pSC101 replication of E. coli.

Figure 3 is a map of the plastid pTH-AL203Coh, abbreviated in the figure: Km, anti-connamycin gene; T7 promoter, T7 promoter; AHL, gene encoding Agrobacterium radiobacter B11291 AHL; CohI, encoding Clostridium thermocellum adhesion region Gene; pSC101, the origin of pSC101 replication in E. coli.

Figure 4 is a map of the plastid pTac-HDTDoCh, abbreviated in the figure: Ap, anti-ampicillin gene; TrxA, a gene encoding TrxA; HDT, a gene encoding Agrobacterium radiobacter B11291 HDT; Doc1, encoding the anchor region of Clostridium thermocellum Gene; ColE1, the origin of ColE1 replication in E. coli.

Figure 5 is a map of the plastid pTac-TrChHDT, abbreviated in the figure: Ap, anti-ampicillin gene; Tac, Tac promoter; TrxA, gene encoding TrxA; HDT, gene encoding Agrobacterium radiobacter B11291 HDT; ChBD, coding Bacillus circulans WL-12 ChBD gene; ColE1, ColE1 replication origin of E. coli.

Figure 6 is a non-original sodium dodecyl sulfate-polyacrylamide electrophoresis gel to illustrate the protein distribution of recombinant strain BL21(DE3)/pETW-A2HCh; wherein, diameter 1: protein standard; diameter 2: not Soluble protein produced by the induced recombinant strain; diameter 3: soluble protein produced by the induced recombinant strain; diameter 4: insoluble protein produced by the uninduced recombinant strain; run 5: insoluble by the induced recombinant strain Protein; arrow: fusion protein of AHL-HDT.

Figure 7 is a diagram showing the non-original sodium dodecyl sulfate-polyacrylamide electrophoresis gel to illustrate the proteins produced by the recombinant strains BAD-5/pTH-ChA203/pTac-TrChHDTh and BAD-5/pTH-AL203Coh/pTac-HDTDoCh. Distribution; wherein, diameter 1: protein standard; diameter 2: soluble protein produced by the induced recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDTh; run 3: induced recombinant strain BAD-5/pTH-AL203Coh Soluble protein produced by /pTac-HDTDoCh; Path 4: Soluble protein produced by induced strain BAD-5 (as a control); arrow from top to bottom in order: TrxA-HDT-Doc1-ChBD fusion protein, TrxA- HDT-ChBD fusion protein, AHL-CohI fusion protein, AHL-ChBD fusion protein.

Figure 8 (A) is a non-associated sodium dodecyl sulfate-polyacrylamide electrophoresis gel to illustrate the adsorption of proteins and chitin spheres produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-TrChHDT; Among them, diameter 1: protein standard; diameter 2: cell extract before adsorption; diameter 3: cell extract after adsorption; diameter 4: cleaning solution after first adsorption after adsorption; diameter 5: after adsorption Washing solution with 2 washes; Path 6: Heating and separating the adsorbed proteins; arrows from top to bottom in order: TrxA-HDT-ChBD fusion protein, AHL-CohI fusion protein.

Figure 8 (B) is a non-primary sodium dodecyl sulfate-polyacrylamide electrophoresis gel to illustrate the adsorption of proteins and chitin spheres produced by the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh; Among them, diameter 1: protein standard; diameter 2: cell extract before adsorption; diameter 3: cell extract after adsorption; diameter 4: cleaning solution after first adsorption after adsorption; diameter 5: after adsorption Washing solution with 2 washes; Path 6: Heating and separating the adsorbed proteins; arrows from top to bottom are: TrxA-HDT-Doc1-ChBD fusion protein, AHL-CohI fusion protein.

Figure 9 (A) is a graph showing the results of D-HPG conversion of the recombinant strain BAD-5/pTH-ChA203/pTac-TrChHDT; wherein the data in the graph were taken from the mean and standard deviation of 3 independent experiments.

Figure 9 (B) is a graph showing the results of D-HPG conversion of the recombinant strain BAD-5/pTH-AL203Coh/pTac-HDTDoCh; wherein the data in the figure were taken from the mean and standard deviation of 3 independent experiments.

<110> Feng Chia University

<120> Method for producing a fusion protein in a cell

<160> 24

<210> 1

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 1

<210> 2

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 2

<210> 3

<211> 2310

<212> DNA

<213> Artificial sequence

<220>

<223> Gene fragment encoding the AHL-HDT fusion protein

<400> 3

<210> 4

<211> 770

<212> PRT

<213> Artificial sequence

<220>

<223> AHL-HDT fusion protein

<400> 4

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 5

<210> 6

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 6

<210> 7

<211> 1104

<212> DNA

<213> Artificial sequence

<220>

<223> Gene fragment encoding the AHL-ChBD fusion protein

<400> 7

<210> 8

<211> 368

<212> PRT

<213> Artificial sequence

<220>

<223> AHL-ChBD fusion protein

<400> 8

<210> 9

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 9

<210> 10

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 10

<210> 11

<211> 1386

<212> DNA

<213> Artificial sequence

<220>

<223> Gene fragment encoding the AHL-CohI fusion protein

<400> 11

<210> 12

<211> 462

<212> PRT

<213> Artificial sequence

<220>

<223> AHL-CohI fusion protein

<400> 12

<210> 13

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 13

<210> 14

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 14

<210> 15

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 15

<210> 16

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 16

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 17

<210> 18

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 18

<210> 19

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 19

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 20

<210> 21

<211> 2301

<212> DNA

<213> Artificial sequence

<220>

<223> Gene fragment encoding the TrxA-HDT-Doc1-ChBD fusion protein

<400> 21

<210> 22

<211> 767

<212> PRT

<213> Artificial sequence

<220>

<223> TrxA-HDT-Doc1-ChBD fusion protein

<400> 22

<210> 23

<211> 2070

<212> DNA

<213> Artificial sequence

<220>

<223> Gene fragment encoding the TrxA-HDT-ChBD fusion protein

<400> 23

<210> 24

<211> 690

<212> PRT

<213> Artificial sequence

<220>

<223> TrxA-HDT-ChBD fusion protein

<400> 24

Claims (14)

A method for producing a fusion protein, comprising: (a) constructing a first nucleic acid molecule encoding an anchor region and a first target peptide, the first target peptide comprising at least a D-type endogenous enzyme (D- Hydantoinase (HDT); (b) constructing a second nucleic acid molecule encoding an adhesion region and a second target peptide, the second target peptide comprising at least D-carbamoylase (AHL) (c) delivering the first nucleic acid molecule and the second nucleic acid molecule into an E. coli; and (d) culturing the E. coli to express a first fusion peptide and a second fusion peptide, and the a fusion peptide having the anchor region and the first target peptide, the second fusion peptide having the adhesion region and the second target peptide, neither the first fusion peptide nor the second fusion peptide An insoluble inclusion body is formed whereby the anchor region is attached to the adhesion region to allow the first fusion peptide and the second fusion peptide to fuse with each other within the E. coli. The method of manufacture of claim 1, wherein the anchoring region is derived from the group consisting of: Acetivibrio cellulolyticus , Bacteroides cellulosolvens , Butyrivibrio fibrisolvens , Clostridium acetobutylicum , Clostridium cellobioparum , Clostridium cellulolyticum , Clostridium cellulovorans , Clostridium Josui , Clostridium papyrosolvens , Clostridium thermocellum , Ruminococcus albus , Ruminococcus flavefaciens , Neocallimastix patriciarum , Orpinomyces joyonii , Orpinomyces PC-2 , Piomyces equi , Piomyces E2 . The manufacturing method according to claim 1, wherein the adhesion region is derived from the group consisting of: Acetivibrio cellulolyticus , Bacteroides cellulosolvens , Butyrivibrio fibrisolvens , Clostridium acetobutylicum , Clostridium cellobioparum , Clostridium cellulolyticum , Clostridium cellulovorans , Clostridium josui , Clostridium papyrosolvens , Clostridium thermocellum , Ruminococcus albus , Ruminococcus flavefaciens , Neocallimastix patriciarum , Orpinomyces joyonii , Orpinomyces PC-2 , Piomyces equi , Piomyces E2 . The manufacturing method according to claim 1, wherein the step (a) comprises implanting the first nucleic acid molecule into a vector for expressing the Escherichia coli, and the step (b) includes the A nucleic acid molecule is implanted into the vector into which the first nucleic acid molecule is implanted. The method of manufacture of claim 4, wherein the vector further has a promoter operably linked to the first nucleic acid molecule and the second nucleic acid molecule. The method of manufacture of claim 4, wherein the vector further comprises a marker gene or a reporter gene for screening the vector. The manufacturing method of claim 1, wherein the step (a) comprises implanting the first nucleic acid molecule into a first carrier for expression by the Escherichia coli, and the step (b) comprises The second nucleic acid molecule is implanted into a second vector that can be expressed by the E. coli. The method of manufacturing of claim 7, wherein the first carrier further has a first promoter operably linked to the first nucleic acid molecule, the second carrier further having an operatively coupled to the A second promoter of the second nucleic acid molecule. The method of manufacture of claim 7, wherein the first vector and the second vector each further comprise a marker gene or reporter gene for screening the vectors. The method of claim 1, wherein the first nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 21 or a degenerate sequence thereof, and the second nucleic acid molecule comprises SEQ ID NO: A nucleic acid sequence or a degenerate sequence thereof. An Escherichia coli comprising: a first nucleic acid molecule encoding an anchor region and a first target peptide, the first target peptide comprising at least a D-type endogenous enzyme; and a second nucleic acid molecule Encoding an adhesion region and a second target peptide, the second target peptide comprising at least a D-formylmethionyl hydrolase; thereby expressing a first fusion peptide and a second fusion in the E. coli a peptide, wherein the first fusion peptide has the anchor region and the first target peptide, the second fusion peptide has the adhesion region and the second target peptide, the first fusion peptide and the second None of the fusion peptides form insoluble inclusion bodies; in turn, the anchor region is attached to the adhesion region to allow the first fusion peptide and the second fusion peptide to fuse with each other in the E. coli. The Escherichia coli according to claim 11, wherein the first nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 21 or a degenerate sequence thereof, and the second nucleic acid molecule comprises SEQ ID NO: A nucleic acid sequence or a degenerate sequence thereof. The Escherichia coli according to claim 11, wherein the anchoring region is derived from the group consisting of: Acetivibrio cellulolyticus , Bacteroides cellulosolvens , Butyrivibrio fibrisolvens , Clostridium acetobutylicum , Clostridium cellobioparum , Clostridium cellulolyticum , Clostridium cellulovorans , Clostridium Josui , Clostridium papyrosolvens , Clostridium thermocellum , Ruminococcus albus , Ruminococcus flavefaciens , Neocallimastix patriciarum , Orpinomyces joyonii , Orpinomyces PC-2 , Piomyces equi , Piomyces E2 . The Escherichia coli according to claim 11, wherein the adhesion region is derived from the group consisting of: Acetivibrio cellulolyticus , Bacteroides cellulosolvens , Butyrivibrio fibrisolvens , Clostridium acetobutylicum , Clostridium cellobioparum , Clostridium cellulolyticum , Clostridium cellulovorans , Clostridium josui , Clostridium papyrosolvens , Clostridium thermocellum , Ruminococcus albus , Ruminococcus flavefaciens , Neocallimastix patriciarum , Orpinomyces joyonii , Orpinomyces PC-2 , Piomyces equi , Piomyces E2 .