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Sequence of high-expression high-specific-activity H1 Collagenase mutant, construction of Pichia pastoris expression plasmid, and methods for screening and purifying strain

A technology of skin fly collagenase and expression plasmid, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and introduction of foreign genetic material using vectors, to achieve the effects of high yield, low production cost, and reduced production cost.

Active Publication Date: 2012-09-12
海南华研胶原科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the collagenase from the larvae of the dermatophyte cannot be produced in large quantities due to the low content in the larvae, complicated extraction process and high production cost, which hinders the popularization and use of collagenase from the dermatophyte

Method used

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  • Sequence of high-expression high-specific-activity H1 Collagenase mutant, construction of Pichia pastoris expression plasmid, and methods for screening and purifying strain

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Experimental program
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Effect test

Embodiment 1

[0032] Construction of Hl Collagenase-m Pichia pastoris engineering strain

[0033] 1. According to the amino acid sequence of the skin fly collagenase mutant and the codon preference of yeast, design the full nucleotide sequence of the skin fly collagenase mutant, and introduce the corresponding enzyme cutting sites XhoI and XbaI at both ends of the sequence, by The sequence was synthesized by Dalian Bao Biological Company;

[0034] 2. Using gene cloning technology, insert the XhoI / XbaI site downstream of the GAP promoter / α-factor signal peptide of pGAPZα-A after XhoI / XbaI double enzyme digestion of the mutant gene of skin fly collagenase to construct a gene containing α-factor Secretory Pichia pastoris expression vector of signal peptide; Hl Collagenase-m / pGAPZα-A expression vector molecular size is 3.759kb, of which pGAPZα-A is 3.147kb, Hl Collagenase-m is 612bp (including terminator), skin fly collagen The enzyme mutant is inserted between the 747bp and 824bp (XbaI site)...

Embodiment 2

[0037] Purification method of collagenase mutant of skinfly

[0038] 1. Take out the Hl Collagenase-m / pGAPZα-A / GS115 engineering bacteria from the -80°C seed bank, and inoculate the plate to activate the strain;

[0039] 2. Pick a single colony from the plate and put it in 200 ml of YPD+zeocin100 mg / L medium, 30°C, 250 rpm, and cultivate for 48 hours. This is the primary seed solution;

[0040] 3. Take 100 ml of primary seed liquid and inoculate it into 2 liters of YPD medium, culture at 30°C, 250 rpm for 24 hours, this is the secondary seed liquid;

[0041] 4. Inoculate 2 liters of secondary seed liquid in a fermenter containing 40 liters of YPD medium, ferment for 72 hours, collect the fermentation supernatant by centrifugation, adjust the pH to 8.0-9.0 with 1 mole of sodium hydroxide solution, pass DEAE SepharoseFF chromatographic column, collect the elution peak of 0.3 mol / L sodium chloride solution, dialyze overnight to remove salt, at this time, the crude product of co...

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Abstract

The invention relates to a sequence of an H1 Collagenase mutant, a key technology for constructing a yeast strain H1 Collagenase-m / pGAPZalpha-A / GS115 for performing secretory expression on the H1 Collagenase mutant, a construction result and a purification method for producing high-activity H1 Collagenase by using the yeast strain. The high-activity H1 Collagenase mutant is produced by a genetic engineering expression method; the specific activity reaches 1,320 IU / mg; the expression level reaches 241 mg / L; target protein, namely H1 Collagenase with the purity of over 95 percent, can be obtained through purification; and a good foundation is laid for the further popularization of the H1 Collagenase.

Description

technical field [0001] The present invention relates to the confirmation of the sequence of Hl Collagenase with a high specific liveness, the construction of the Pichia expression vector of the sequence, and the secretion and expression of the Pichia strain Hl Collagenase-m / pGAPZα-A / GS115 The key technology of the construction, the result of the construction and the purification method of using it to produce highly active collagenase. The invention belongs to the field of biotechnology. Background technique [0002] The chemical name of collagenase is collagenase (Collagenase), which can specifically hydrolyze the three-dimensional helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. Collagenase from Clostridium cleaves amino-terminal peptide bonds of glycine residues in collagen. Collagenase from Hypoderma lineatum larvae cleaves the amino-terminal peptide bond of alanine residues in native ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/63C12N15/66C12N1/19C12R1/84
Inventor 陈海宁程云开
Owner 海南华研胶原科技股份有限公司