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Fusion protein CR2-Linker-GDH and application thereof

A fusion protein and sequence technology, which is applied in the direction of fusion polypeptide, recombinant DNA technology, and the use of vectors to introduce foreign genetic material, etc., can solve the problems of high cost, troublesome operation, and low efficiency of coenzyme regeneration

Inactive Publication Date: 2015-03-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the traditional enzyme coupling regeneration system, two enzymes, the substrate catalytic enzyme and the coenzyme recycling enzyme, need to be added separately. The operation is troublesome and the cost is high, and the coenzyme is transferred between the two enzyme molecules, which may result in low coenzyme regeneration efficiency.

Method used

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  • Fusion protein CR2-Linker-GDH and application thereof
  • Fusion protein CR2-Linker-GDH and application thereof
  • Fusion protein CR2-Linker-GDH and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the acquisition of fusion gene

[0027] The cores of carbonyl reductase CR2 and glucose dehydrogenase GDH were obtained from E.coliBL21 / pET-CR2 and E.coliBL21 / pET-GDH by extracting plasmids, PCR amplification, or direct chemical synthesis. Nucleotide sequence (respectively shown in SEQ ID NO.5, SEQ ID NO.6), its amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.2 respectively.

[0028] A flexible oligopeptide linker (GGGGS) was added between the two genes using overlap extension PCR 3 , get the fusion expression gene cr2-linker-gdh. The nucleotide sequence of the fusion gene is shown in SEQ ID NO.4, and the encoded amino acid sequence is shown in SEQ ID NO.3.

[0029] The method for obtaining the fusion gene is specifically:

[0030] (1) Using the plasmid pET-cr2 as a template, using upstream primer 1 (sequence shown in SEQ ID NO.7) and downstream primer 2 (sequence shown in SEQ ID NO.8), amplify the cr2 gene fragment by PCR reaction ;

[0031] (...

Embodiment 2

[0033] Embodiment 2: Construction of the genetically engineered bacteria expressing fusion protein CR2-Linker-GDH

[0034] (1) After the fusion gene obtained in Example 1 is purified, it is connected to the pMD-19T carrier, transformed into Escherichia coli E.coliJM109 competent cells, and the correct recombinant plasmid pMD-C-L-G is screened, and verified by sequencing;

[0035] (2) Digest the recombinant plasmids pMD-C-L-G and pET-28a with restriction endonucleases Nde Ⅰ and Not Ⅰ respectively, recover and purify the digested products, and linearize the pET-28a vector and c-l-g gene fragments in T4 Ligated under the action of ligase to obtain a recombinant plasmid with two target genes cr2 and gdh, screened the correct recombinant plasmid and named it pET-C-L-G.

[0036] (3) Transform the competent E.coliBL21(DE3) with the recombinant plasmid pET-C-L-G obtained in the previous step to obtain the final positive strain E.coli BL21 / pET-C-L-G.

Embodiment 3

[0037] Embodiment 3: Induced expression of fusion protein

[0038]Pick a single colony of the recombinant strain E.coli BL21 / pET-C-L-G and inoculate it in 5 mL of LB liquid medium containing 50 μg / mL kanamycin, culture it overnight at 37°C with shaking at 200 rpm, and transfer 1 mL of the culture liquid to 50 ml In the LB liquid medium containing 50μg / mL kanamycin, culture at 37°C and 200rpm with shaking until the OD is 0.6-0.8, then add the inducer isopropyl-D-thiogalactoside IPTG 0~ 1mmol / L, induced culture at 17°C for 10h; centrifuge at 10,000rpm for 10min, collect the bacteria, wash twice with saline, and collect the whole cells of the recombinant bacteria. The expression of fusion protein can reach 0.143g / g (protein / cell)

[0039] Resuspend the collected whole cells of the recombinant bacteria in the injection buffer used for purification, and ultrasonically disrupt: working time 2s, intermittent time 3s, 20min in total; centrifuge the disrupted solution at 12000rpm for ...

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Abstract

The invention discloses a fusion protein CR2-Linker-GDH and the application of the fusion protein CR2-Linker-GDH, and belongs to the technical field of genetic engineering and biological catalysis. Fusion expression is carried out on carbonyl reductase and glucose dehydrogenase to achieve coupling between the two enzymes, and a separated and purified fusion protein in a successfully constructed recombinant bacteria package is utilized to carry out biotransformation COBE to obtain S-CHBE. By optimizing reaction conditions, a substrate of the fusion protein can be totally converted, the optical purity of a product is above 99%, and the TTN reaches up to 1200. The final concentration of the substrate is 305 mmol / L through a substrate batch replenishing strategy, the yield of the product reaches up to 53%, the product of 162 mmol / L is obtained, and the e.e. value remains above 99%. The fusion protein CR2-Linker-GDH and the application of the fusion protein CR2-Linker-GDH provide a novel research thought for coupling a coenzyme regeneration cycle approach into a biotransformation approach of the S-CHBE and have great significance for simplifying the a chiral transformed way.

Description

technical field [0001] The invention relates to a fusion protein CR2-Linker-GDH and an application thereof, belonging to the technical fields of genetic engineering and biocatalysis. Background technique [0002] Chiral alcohol compounds are important intermediates in the fields of medicine / food / pesticide chemistry / fine chemicals / cosmetics. Optically pure (S)-4-chloro-3-hydroxybutyrate ethyl ester [(S)-CHBE] is a synthetic cholesterol-lowering drug Atovastatin (Atovastatin) / hydroxymethylglutaric acid formyl-CoA (HMG -CoA) Reductase inhibitors and precursors of drugs such as alkaloids SlageninsB and C. In its production method, the biological method has the advantages of mild reaction conditions / less reaction steps, and the method has strong stereo and enantioselectivity. [0003] The theoretical yield of biotransformation by oxidoreductase can reach 100%, but in the application of oxidoreductase, in addition to the need for suitable enzymes, it is also necessary to provi...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N9/04C12N15/70C12N1/21C12P7/62
CPCC12N9/0006C07K2319/00C12P7/62C12Y101/01184C12Y101/9901
Inventor 穆晓清徐岩
Owner JIANGNAN UNIV
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