Fructosamine deglycase pichia pastoris expression vector, genetically engineered bacterium, construction method and protein expression method

A technology of fructosamine desugarase and genetically engineered bacteria, which is applied in the field of protein expression, can solve problems such as unfavorable large-scale industrial production, and achieve the effects of convenient large-scale production, no environmental pollution, and simple operation

Pending Publication Date: 2021-10-29
WUHAN BAIANHUIJI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Fructosamine desugarase is currently constructed in Escherichia coli, which is not conducive to the large-scale industrial production of this enzyme in the field of food and drug

Method used

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  • Fructosamine deglycase pichia pastoris expression vector, genetically engineered bacterium, construction method and protein expression method
  • Fructosamine deglycase pichia pastoris expression vector, genetically engineered bacterium, construction method and protein expression method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Construction of yeast expression vector of fructosamine desugarase gene

[0047] (1) Primer design: design primers starting from the mature peptide sequence after the signal peptide (as shown in SEQ ID NO.3 and SEQ ID NO.4);

[0048] (2) PCR reaction, using the cloning vector pColdII-FrIB as a template (the present invention has no limitation on the construction method of pColdII-FrIB, and conventional construction methods can be used to construct FrIB in pColdII-FrIB), annealing at 62°C, 35 cycles.

[0049] (3) The PCR product of the corresponding biological enzyme gene digested by SnaBI or NotI and the plasmid pPIC9K.

[0050] Table 1 Double enzyme digestion system

[0051] Element Usage amount Purification of PCR products / plasmids 30μL 10*quitcut buffer 5μL QuitCut SnaBI 1μL Quit Cut Not I 1μL wxya 2 o

13μL total capacity 50μL

[0052] Enzyme digestion at 37°C for 2h.

[0053] (4) Ligate, transform, and...

Embodiment 2

[0056] Construction and Induced Expression of Recombinant Yeast Containing Fructosamine Desugarase Gene

[0057] 1. Preparation of Pichia pastoris electroporation competent

[0058] Inoculate a single colony of Pichia pastoris GS115 into 25mL YPD medium, cultivate overnight at 30°C, 250r / min to OD 600 About 2 to 6. Transfer 1mL culture to 100mLYPD medium, continue to culture to OD 600 About 1.4. 6,000r / min, after centrifugation for 10min, discard the supernatant. The cells were gently resuspended in 50mL Solution I solution, and after standing at room temperature for 30min, centrifuged at 6,000r / min at 4°C for 10min. After discarding the supernatant, gently resuspend the cells in 10mL 1mol / L sorbitol solution, wash the cells 4 times, and gently resuspend the cells in 0.5mL 1mol / L sorbitol solution again, aliquot 80μL per tube , and stored in a -80°C refrigerator for later use.

[0059] 2. Linearization of recombinant plasmid pPIC9K-FrIB

[0060] Linearize recombinant pla...

Embodiment 3

[0067] Purification of fructosamine desugarase protein

[0068] Purification of recombinant enzyme and its analysis by SDS-PAGE gel electrophoresis

[0069] For protein purification, refer to GE Healthcare guidelines, and for SDS-PAGE analysis, follow the "Molecular Cloning Experiment Guide" (Third Edition), the gel concentration used is 12.5%, and the loading volume is 5-25 μL. Proteins were stained with Coomassie brilliant blue R-250.

[0070] Among them, native-SDS-PAGE experimental steps:

[0071] A. Add 5-10 μL sample buffer solution [0.1mol / L Tris-HCl (Tris-HC1), pH 6.8; 2% SDS (weight: volume), 10% glycerol ( volume: volume), 0.01% bromophenol blue (weight: volume)] placed in a water bath at 37°C for 5-10 min, and then separated by electrophoresis. Note: When extracting samples, the purpose of not adding mercaptoethanol to the sample extract is to moderately denature proteases during electrophoresis, so that the activity of these proteases can be restored after electro...

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Abstract

The invention relates to a fructosamine deglycase pichia pastoris expression vector, a genetically engineered bacterium, a construction method and a protein expression method, and belongs to the technical field of protein expression. The fructosamine deglycase pichia pastoris expression vector disclosed by the invention comprises a skeleton vector and a gene for coding fructosamine deglycase; and the nucleotide of the gene for coding the fructosamine deglycase is as shown in SEQ ID NO. 1. The vector disclosed by the invention is beneficial to industrial production of the fructosamine deglycase, is green in production method, safe, environment-friendly, pollution-free and controllable, is beneficial to large-scale preparation of the glucosamine, and can generate greater social value and economic benefits.

Description

technical field [0001] The invention relates to the technical field of protein expression, in particular to an expression vector of fructosamine desugarase Pichia pastoris, a genetic engineering bacterium, a construction method and a protein expression method. Background technique [0002] Glucosamine (Glucosamine, GlcN), namely 2-amino-2-deoxy-D-glucose, also known as glucosamine, glucosamine or simply glucosamine, ammonia sugar, is a compound in which a hydroxyl group of glucose is replaced by an amino group . Studies have shown that glucosamine has some special physiologically active functions, such as anti-inflammatory and analgesic, repairing cartilage damage, treating rheumatoid arthritis; strengthening white blood cell proliferation and differentiation, promoting cytokine secretion, improving immune regulation, anti-tumor; restoring mitochondrial enzymes Expression, improve mitochondrial glutathione antioxidant capacity, enhance immune function; promote endoplasmic r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66C12N15/55C12N1/19C12N9/78C12R1/84
CPCC12N15/815C12N9/78
Inventor 王怀英
Owner WUHAN BAIANHUIJI BIOTECHNOLOGY CO LTD
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