CN102660524A - Sequence of high-expression high-specific-activity H1 Collagenase mutant, construction of Pichia pastoris expression plasmid, and methods for screening and purifying strain - Google Patents

Abstract

The present invention relates to the sequence of the mutant HlCollagenase and the key technology for the construction of the secreted and expressed yeast strain HlCollagenase-m/pGAPZα-A/GS115, the construction results and the purification of HlCollagenase produced by using it method. The present invention adopts the method of genetic engineering expression to produce highly active skin fly collagenase mutant, the specific activity reaches 1320 international units/mg, the expression amount reaches 241 mg/liter protein, and the target protein skin with a purity of more than 95% can be obtained through purification. fly collagenase, laying a good foundation for the promotion and use of skin fly collagenase in the future.

Description

High-expression high specific live skin fly collagenase mutant sequence, construction of Pichia pastoris expression plasmid, strain screening and purification method

technical field

The present invention relates to the confirmation of the sequence of collagenase (Hl Collagenase) with a high specific liveness, the construction of the Pichia pastoris expression vector of the sequence, and the Pichia yeast strain H1 Collagenase-m/pGAPZα-A/GS115 secreting and expressing the fly collagenase 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

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 the larvae of the skin fly (Hypoderma lineatum) cleaves the amino-terminal peptide bond of alanine residues in native collagen. Collagenase widely used in medical treatment is extracted and prepared from Clostridium histolyticum, which mainly hydrolyzes collagen components in connective tissue. However, the collagenase from the larvae of the skin fly cannot be mass-produced so far due to the low content in the body of the larvae, complicated extraction process and high production cost, which hinders the popularization and use of the collagenase from the skin fly.

Contents of the invention

Derma collagenase is the main component of the soluble antigen of the first-stage larva of Derma larvae. It is a serine protease with a molecular weight of 25223 Daltons. The mature Derma collagenase contains 230 amino acids, which is 30 amino acids less than its precursor. The whole amino acid sequence has a high similarity with that of the trypsin family serine proteases. The active functional fragment is 45-180 amino acids, and the substrate-binding region is 174-201 amino acids; the whole sequence has 6 cysteines (30,46; 151,166; 176,204 amino acids), forming 3 pairs of disulfide bonds. According to the analysis of the molecular structure and sequence of the collagenase of the fly, we designed to excise the amino acid sequence of positions 1-27, so that the functional area can be exposed in a wider range, so that it can better bind to the reaction substrate, thereby improving the activity of the enzyme . And the 29th tryptophan (Trp) was mutated to isoleucine (Ile) to maintain the stability of the molecular structure;

The sequence of the mutant collagenase with high activity is as follows:

Val Ile

The characteristic of this sequence is that only the functional region and substrate binding region (28~230 amino acids) in the mature skin fly collagenase sequence are retained and the tryptophan at position 29 is mutated into leucine. This change is theoretically On the one hand, the functional area is exposed in a wider range, so that it can better combine with the reaction substrate, thereby improving the activity of the enzyme;

At the same time, the present invention adopts the secretory expression system of Pichia pastoris, which has the advantages of high yield, low production cost and post-translational modification function of the eukaryotic expression system, and can be used for mass production of collagenase of the fly, reducing production cost;

The construction method of Pichia pichia pichia collagenase recombinant strain is as follows: insert the modified and mutated skin fly collagenase Hl Collagenase gene into the GAP promoter/α-factor signal peptide downstream site of the pGAPZα-A empty vector, and delete the The Ste13 site Glu-Ala-Glu-Ala after the Kex2 site Lys-Arg was constructed into the expression vector Hl Collagenase-m/ pGAPZα-A of the fly collagenase gene secretion; the Collagenase-m/ pGAPZα -A vector transformed Pichia pastoris ( Pichia pastoris ) strain GS115, and constructed Pichia pastoris engineering strain --- Hl Collagenase-m/ pGAPZα-A/GS115, which secretes and expresses a mutant of pichia collagenase; through antibiotic selection, SDS-PAGE electrophoresis analysis and biological specific activity determination, obtained a recombinant strain of pichia pichia pastoris with high expression and high activity of collagenase mutant;

The target protein of the above-mentioned Pichia pastoris recombinant strain: the expression of Hl Collagenase-m mutant (Hl Collagenase-m) reached 241 mg/L, and the specific activity was 1320 IU/mg; The specific activity of skin fly collagenase is 5~6 times higher, and it has high stability;

The purification method of the skin fly collagenase mutant is as follows: use the highly expressed and high activity skin fly collagenase mutant Pichia pastoris recombinant strain to ferment, centrifuge to collect the supernatant, adjust the pH value with sodium hydroxide, and pass through an anion exchange layer Analyze the column, collect the target elution peak, and obtain the crude product of collagenase mutant with a purity of about 70%, dialyze to remove salt, concentrate by ultrafiltration, pass through a molecular sieve chromatography column, collect the target protein, and obtain collagenase with a purity of more than 95% Enzyme mutant protein.

【Description of drawings】

The attached picture shows the construction of the expression vector of the skin fly collagenase mutant (Hl Collagenase-m)

The molecular size of Hl Collagenase-m/ pGAPZα-A expression vector is 3.759kb. Among them, pGAPZα-A is 3.147kb, H1 Collagenase-m is 612bp (including the terminator code); H1 Collagenase-m is inserted between the 747th and 824bp (XbaI site) of the vector pGAPZα-A;

The structure of the pGAPZα-A vector is:

Phosphoglycerate dehydrogenase gene (GAP) promoter region: 1-483bp

Phosphoglycerate dehydrogenase gene promoter primer site: 455-476bp

α-mating factor secretion signal peptide sequence: 493-759bp

α-mating factor secretion signal peptide primer site: 696-716bp

Vector multiple cloning site: 760-828bp

Myc epitope package: bp 827-856bp

3'-Alcohol oxidase 1 gene primer site: 974-994bp

Alcohol oxidase 1 transcription termination region: 593-1233 bp

Transcription elongation factor 1 promoter region: 1234-1644 bp

Synthetic prokaryotic promoter: bp 1645-1712

Streptomyces zeocin resistance gene reading frame: 1713-2087bp

Cytochrome synthase 1 transcription termination region: bp 2088-2405

E. coli replicon 1 (derived from pUC vector): 2416-3089 bp.

【Detailed ways】

The specific application of the present invention in the construction of the pichia collagenase mutant recombinant Pichia pastoris engineering strain and the purification process of the pichia collagenase mutant is described in detail below in conjunction with the examples;

Example 1

Construction of Hl Collagenase-m Pichia pastoris engineering strain

1. According to the amino acid sequence of the skin fly collagenase mutant and the yeast's preference for codons, the full nucleotide sequence of the skin fly collagenase mutant was designed, and the corresponding restriction sites XhoI and XbaI were introduced at both ends of the sequence. The sequence was synthesized by Dalian Bao Biological Company;

2. Using gene cloning technology, the mutant gene of the collagenase of the fly was inserted into the XhoI/XbaI site downstream of the GAP promoter/α-factor signal peptide of pGAPZα-A after double digestion with XhoI/XbaI 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) of the vector pGAPZα-A, see the attached figure for details;

3. Through the electroporation method, the linearized Hl Collagenase-m/ pGAPZα-A plasmid was transferred into Pichia pastoris GS115 competent (purchased from Invitrogen Company), and positive strains were obtained by screening with zeocin antibiotics, and further PCR and Southern blotting were used to obtain positive strains. The positive strains screened out by technical identification, and then the H1 Collagenase-m/pGAPZα-A/GS115 engineering strain with high expression and high activity was screened by SDS-PAGE electrophoresis method and SDS-PAGE activity assay method;

Example 2

Purification method of collagenase mutant of skinfly

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;

2. Pick a single colony from the plate and put it in 200ml YPD+zeocin100mg/L medium, 30℃, 250 rpm, and cultivate for 48 hours. This is the first-class seed solution;

3. Take 100 ml of the primary seed liquid and inoculate it in 2 liters of YPD medium, at 30°C, 250 rpm, and cultivate for 24 hours. This is the secondary seed liquid;

4. Inoculate 2 liters of secondary seed liquid into 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, and 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 collagenase mutant protein of skin fly with a purity of about 70% can be obtained, concentrated by ultrafiltration, and passed through the G25 layer Analyze the column, collect the peak of the target protein, and obtain the stock solution of the collagenase mutant protein with a purity greater than 95%, and freeze-dry the stock solution for storage. the

<110> Hainan Huayan Biotechnology Co., Ltd.

<120> Construction of Highly Expressed Collagenase Mutant Sequence and Pichia Pichia Expression Plasmid, Strain Screening and Purification

<160> 1

<170> PatentIn Version 2.1

<210> 1

<211> 203

<212> amino acid

<213> Hypoderma lineatum (early cattle grub)

<220>

<221> misc_difference

<400> 1

Val Ile Cys Gly Gly Ser Leu Ile Asp Asn Lys Trp Ile Leu Thr Ala

1 5 10 15

Ala His Cys Val His Asp Ala Val Ser Val Val Val Tyr Leu Gly Ser

20 25 30

Ala Val Gln Tyr Glu Gly Glu Ala Val Val Asn Ser Glu Arg Ile Ile

35 40 45

Ser His Ser Met Phe Asn Pro Asp Thr Tyr Leu Asn Asp Val Ala Leu

50 55 60

Ile Lys Ile Pro His Val Glu Tyr Thr Asp Asn Ile Gln Pro Ile Arg

65 70 75 80

Leu Pro Ser Gly Glu Glu Leu Asn Asn Lys Phe Glu Asn Ile Trp Ala

85 90 95

Thr Val Ser Gly Trp Gly Gln Ser Asn Thr Asp Thr Val Ile Leu Gln

100 105 110

Tyr Thr Tyr Asn Leu Val Ile Asp Asn Asp Arg Cys Ala Gln Glu Tyr

115 120 125

Pro Pro Gly Ile Ile Val Glu Ser Thr Ile Cys Gly Asp Thr Ser Asp

130 135 140

Gly Lys Ser Pro Cys Phe Gly Asp Ser Gly Gly Pro Phe Val Leu Ser

145 150 155 160

Asp Lys Asn Leu Leu Ile Gly Val Val Ser Phe Val Ser Gly Ala Gly

165 170 175

Cys Glu Ser Gly Lys Pro Val Gly Phe Ser Arg Val Thr Ser Tyr Met

180 185 190

Asp Trp Ile Gln Gln Asn Thr Gly Ile Lys Phe

195 200 203

the

the

Claims (8)

1. a torsalo collagenase (Hl Collagenase) two mutants specifically has following sequence

Val Ile CysGlyGlySerLeuIleAspAsnLysTrpIleLeuThrAlaAlaHisCysValHisAspAlaValSerValValValTyrLeuGlySerAlaValGlnTyrGluGlyGluAlaValValAsnSerGluArgIleIleSerHisSerMetPheAsnProAspThrTyrLeuAsnAspValAlaLeuIleLysIleProHisValGluTyrThrAspAsnIleGlnProIleArgLeuProSerGlyGluGluLeuAsnAsnLysPheGluAsnIleTrpAlaThrValSerGlyTrpGlyGlnSerAsnThrAspThrValIleLeuGlnTyrThrTyrAsnLeuValIleAspAsnAspArgCysAlaGlnGluTyrProProGlyIleIleValGluSerThrIleCysGlyAspThrSerAspGlyLysSerProCysPheGlyAspSerGlyGlyProPheValLeuSerAspLysAsnLeuLeuIleGlyValValSerPheValSerGlyAlaGlyCysGluSerGlyLysProValGlyPheSerArgValThrSerTyrMetAspTrpIleGlnGlnAsnThrGlyIleLysPhe。

2. a torsalo collagenase two mutants expression vector is characterized in that having changed in the expression plasmid the described torsalo collagenase of claim 1 mutant gene.

3. torsalo collagenase two mutants expression vector according to claim 2 is characterized in that described expression plasmid pGAPZ α-A.

4. the structure of a torsalo collagenase two mutants expression vector is characterized in that the torsalo collagenase Hl Collagenase gene of modifying sudden change is inserted the GAP promotor/α-site, factor signal peptide downstream of pGAPZ α-A empty carrier, and deletes on the carrier Kex2After the Lys-Arg of site Ste13Site Glu-Ala-Glu-Ala is built into torsalo collagenase gene secreted expression carrier Hl Collagenase-m/ pGAPZ α-A.

5. a transformant is characterized in that the host bacterium is transformed by claim 2 or 3 described torsalo collagenase expression carriers.

6. transformant according to claim 5, it is characterized in that described host bacterium be pichia spp ( Pichia pastoris) bacterial strain GS115.

7. a method of producing torsalo collagenase two mutants is characterized in that cultivating claim 5 or 6 described transformants, collects its secretory product.

8. the purification process of a torsalo collagenase two mutants Pichia anomala expression product is characterized in that directly separation and purification torsalo collagenase two mutants from the fermented liquid of secretor type torsalo collagenase two mutants Pichi strain Hl Collagenase-m/ pGAPZ α-A/GS115: centrifugal collection supernatant, regulate pH value with sodium hydroxide; Cross the anion-exchange chromatography post; Collect the target elution peak, obtain the torsalo collagenase two mutants bullion of purity about 70%, the dialysis desalination; Ultrafiltration and concentration; Cross molecular sieve chromatography, collect target protein, obtain purity greater than 95% torsalo collagenase mutant protein.