CN106191016A - A kind of preparation method of recombinant lysine specific enzyme - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, provides a preparation method of a recombinant lysine specific enzyme, and relates to design of a gene sequence of the recombinant lysine specific enzyme, construction of a recombinant expression vector and a genetic engineering bacterium containing the gene, and a preparation method of the recombinant lysine specific enzyme. The method overcomes the defects of low production efficiency, long fermentation period, complex extraction process and the like in the prior art, has the characteristics of rapidness, simplicity, convenience, stability and the like while reducing the production cost, and the produced rLys-C has high activity, provides an effective way for the industrial production of the recombinant lysine specific enzyme, and has important application and research values for the development of genetic engineering medicines and biological research.

Description

A kind of preparation method of recombinant lysine specific enzyme

technical field

The invention relates to the technical field of genetic engineering. Specifically, the present invention provides a high-efficiency preparation method of recombinant lysine-specific endoprotease (Lys-C), including the construction of engineering bacteria, expression and purification of recombinant lysine-specific enzyme.

Background technique

Lysine-specific endoprotease is an alkaline protease with a molecular weight of about 26kD and belongs to the serine protease family. As a high-purity protease, it can specifically hydrolyze the carboxyl terminus of Lys (except when linked to arginine), and can generate more enzymatic peptides than Trypsin, which can be used for peptide mass fingerprinting or MS/MS spectral matching. Therefore, it has a good application prospect in proteomics analysis. In addition, because of its high specificity and high enzyme cleavage activity, Lys-C can also be used for precursor cleavage (such as insulin precursor) in the industrial production of recombinant proteins, so Lys-C is currently a protein expression tool in the field of genetic engineering pharmaceuticals An important tool enzyme for downstream purification, especially suitable for bioengineering pharmaceutical industry and genetic engineering, biochemistry, molecular biology research.

Most of the lysine-specific enzymes currently on the market are the expression products of natural strains. It is known that three microorganisms can synthesize Lys-C: Lysobacillus enzymogenes, Achromobacter hydrolyzate, and Pseudomonas aeruginosa. Lysine-specific enzymes are mainly expressed and produced by Lysobacterium enzymogenes, followed by isolation from the culture solution of Achromobacter hydrolyzate. Naturally extracted Lys-C is extremely expensive, for example, the market price of Promega's Lys-C is about 370 yuan/μg. At the same time, as the main producer of Lys-C, Lysobacterium enzymogenes has low production efficiency, long fermentation period, and complicated extraction process, which greatly limits the wide application of Lys-C.

At present, the naturally extracted lysine-specific enzymes are not only limited, but also have high cost and low yield, and are easily contaminated by other impurities, which will lead to the degradation of the target protein. At the same time, there is currently no domestic enterprise for the recombinant production of lysine-specific enzymes, and the price of imported rLys-c is relatively expensive. Purchasing imported rLys-c in large quantities will greatly increase the cost of drug production. Therefore, establishing a fast, simple and stable , The production method of rLys-C with high activity has important application and research value for the development of genetic engineering drugs and biological research.

Contents of the invention

In order to overcome the defects and deficiencies of the above-mentioned prior art, the present invention provides a method for preparing a recombinant lysine-specific enzyme, which involves the design of a recombinant lysine-specific enzyme gene sequence, a recombinant expression vector containing the gene and genetic engineering The construction of bacteria and the preparation method of recombinant lysine specific enzyme. An effective way is provided for the industrial production of the recombinant lysine-specific enzyme.

One aspect of the present invention provides a high-efficiency expression recombinant lysine-specific enzyme, the DNA sequence of which is SEQ ID NO:1.

The amino acid sequence of the above-mentioned recombinant lysine-specific enzyme is SEQ ID NO:2.

A recombinant vector containing pPIC9k-PIV with the sequence of SEQ ID NO:1.

On the other hand, the technical solution of the present invention provides an engineering bacterium for highly expressing a recombinant lysine-specific enzyme, and the engineering bacterium is Pichia pastoris containing the sequence SEQ ID NO:1.

In some embodiments of the present invention, the engineering bacteria is GS115/pPIC9k-PIV.

The technical solution of the present invention also provides a method for constructing highly efficient recombinant lysine-specific enzyme engineering bacteria, comprising the following steps:

1) Obtain the optimized DNA sequence SEQ ID NO: 1, add a 6×His tag to its C-terminus, and add appropriate enzyme cutting sites and corresponding Kex2 sites to the 5' and 3' ends of the gene sequence, and finally carry out a complete Gene synthesis to obtain PIV;

2) inserting the PIV connected with the leader peptide α factor into the plasmid pPIC9k to obtain the recombinant plasmid pPIC9k-PIV;

3) The plasmid pPIC9k-PIV obtained above was subjected to Sac linearization, and electrotransformed into Pichia pastoris GS115 strain, and the high-yielding strain GS115/pPIC9k-PIV was screened through cultivation and enzyme activity detection.

A method for producing recombinant lysine-specific enzymes using the above-mentioned genetically engineered bacteria comprises the following steps of cultivating genetically engineered bacteria and obtaining recombinantly expressed recombinant lysine-specific enzymes.

A method for producing recombinant lysine-specific enzymes using the above-mentioned genetically engineered bacteria, characterized in that it comprises the following steps:

a) Screening of high-yielding strains of lysine-specific enzymes: Inoculate the strains into the primary medium, cultivate them at 28-30°C until the OD ₆₀₀ is 15-20, and then transfer them to the secondary medium, 28-30 Shake culture at ℃, add methanol every 8-12h to induce expression, stop fermentation after 12-120h, centrifuge the culture medium to get the supernatant;

b) Fermentation production: inoculate the re-screened bacterial strains into the fermentation medium, cultivate at 28-30°C until the dissolved oxygen reaches 70-80%, add glycerol at 2-4ml/min for 9-12 hours, add The methanol solution containing 1% to 1.2% PTM1 is used for induction, and after 60-72 hours, the fermentation broth is taken every 4-12 hours for enzyme activity detection;

c) Separating and purifying the above-mentioned culture solution to obtain a lyophilized protein powder of the lysine-specific enzyme protein.

In some embodiments of the present invention, the inoculum amount of the strain in the fermentation medium in step b) is 1-5%.

In some embodiments of the present invention, the pH value of the medium during methanol induction in step a) is 4.0-8.0; the pH value of the medium during methanol induction in step b) is 5.5-6.0.

In some embodiments of the invention, purification is selected from exchange chromatography, ultrafiltration or lyophilization.

All ranges cited herein are inclusive of endpoints unless expressly stated to the contrary. For example, "the pH value during methanol induction in step a) is 4.0-8.0" means that the pH value during methanol induction in step a) is 4.0≤pH≤8.0.

Numerals herein are approximate, regardless of whether words such as "about" or "approximately" are used. Numerical values may vary by 1%, 2%, 5%, 7%, 8%, 10%. Whenever a number with a value of N is disclosed, any number with N+/-1%, N+/-2%, N+/-3%, N+/-5%, N+/-7%, N+/-8%, or N+ Figures for /-10% values are explicitly disclosed, where "+/-" means plus or minus.

The invention provides an effective way for the industrial production of the recombinant lysine-specific enzyme after improving the gene of the recombinant lysine-specific enzyme and adopting the expression product of Pichia pastoris. Compared with the currently commonly used expression products of Lysobacterium enzymolyticus, Achromobacter hydrolyzate and Pseudomonas aeruginosa, it overcomes the shortcomings of low production efficiency, long fermentation cycle, and complicated extraction process, and reduces production costs while reducing production costs. It also has the characteristics of fast, simple, stable, etc., and the rLys-C produced has high activity, and has important application and research value for the development of genetic engineering drugs and biological research.

Description of drawings

Figure 1 is a schematic diagram of the recombinant expression plasmid pPIC9k-PIV;

Fig. 2 is a schematic diagram of the enzyme activity assay results of the fermentation broth supernatant at different induction times of PIV;

detailed description

The present invention will be described in further detail below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto, and the process parameters not specifically indicated can be carried out with reference to conventional techniques.

Example 1 Construction of recombinant expression plasmid pPIC9k-PIV

1. Total synthesis of recombinant lysine-specific enzyme gene (PIV)

According to the lysine-specific enzyme gene sequence (AY062882) and amino acid sequence (AAL47683.1) published by GenBank, the lysine-specific enzyme nucleic acid sequence was optimized according to the yeast codon optimization strategy, and 6 was added to its C-terminus. ×His tag, and at the 5' end of the PIV gene sequence, there is an XhoI site and a codon AAAAGA corresponding to the Kex2 protease cleavage site Lys-Arg, and a TGA stop codon and a NotI site are added at the 3' end to obtain the coding gene PIV , and finally handed over to the gene synthesis company for the whole gene synthesis (pUC57-PIV).

2. Construction of recombinant expression plasmid pPIC9k-PIV

The intermediate vector 18T-α treated with XhoI and NotI sites was used as the carrier fragment, which was ligated with the fragment recovered from pUC57-PIV after double digestion with XhoI and NotI, and the positive plasmid 18T-α-PIV was screened. After the obtained positive plasmid 18T-α-PIV was digested with BamHI and NotI, the target gene fragment (α-PIV) was recovered, and connected with the vector fragment recovered after pPIC9k was digested with BamHI and NotI, and the final recombinant expression was obtained by screening Plasmid pPIC9k-PIV, the results are shown in Figure 1. Using 5'AOX1 and 3'AOX1 primers for sequencing, the sequencing results showed that the cloned gene fragment was consistent with the theory.

Example 2 Pichia pastoris GS115 produces lysine specific enzyme

1. pPIC9k-PIV electroporation

The expression plasmid pPIC9k-PIV was linearized using the SacI restriction site, and the competent yeast host strain was prepared according to the method in the EasySele Pichia Expression Kit of Invitrogen Company, and electroporated into the Pichia GS115 strain, and spread on the MD plate. After 3-4 days of culture at 30°C, a single colony was grown.

2. Screening of high-producing strains of lysine-specific enzymes

Select a moderately sized and relatively plump single colony and inoculate it into BMGY medium (mass percentage, yeast powder 1%, peptone 2%, YNB 1.34%, Glycerol 1%, biotin 0.004%, 100mM phosphate buffer), overnight at 30°C Culture, OD ₆₀₀ is about 15-20, transfer to 500mL cone containing 50mL BMMY medium (mass percentage, yeast powder 1%, peptone 2%, YNB1.34%, biotin 0.004%, 100mM phosphate buffer saline) Shaped flasks were cultured at 30°C in a shaker at 220 rpm, and 1% volume of methanol was added every 12 hours to induce expression, and co-cultivated for 96 hours. After the fermentation, the culture medium was taken, centrifuged (12000rpm, 5min, 4°C), and the supernatant was taken. The enzyme activity of the fermentation broth of different single colonies was measured, and the strains with higher enzyme activity were screened, and then the second round of re-screening was carried out.

3. Fermentative production of lysine-specific enzymes

The specific method is as follows: ferment in a 50L fermenter with the high-yield strain obtained by re-screening (the enzyme activity of shake flask fermentation is 0.817U/mL). The inoculum amount of the seed solution was 5%, and transferred to 20L basal medium (glycerol 40g/L, K ₂ SO ₄ 17.5g/L, MgSO ₄ 14g/L, KOH 3.2g/L, CaSO 0.8g/L, PTM1 3ml/L, pH 6.0) in a 50L fermenter, cultivate for 16-18h, wait for the dissolved oxygen to rise from close to 0% to 70-80%, add glycerol at 2mL/min for 10 hours, start induction, optimize the induced pH 5.5-6.0, adding methanol containing 1.2% PTM1 during the stable induction period for 60-72h induction, and co-fermented for 121h, the fermentation broth was taken every 4h for enzyme activity detection, the results are shown in Figure 2.

Embodiment 3 Purification and activity detection of lysine-specific enzyme

1. Purification of Lysine-Specific Enzymes

The supernatant of the fermentation broth was replaced with buffer by an ultrafiltration system. The replacement solution was 30mMTris-HCl. The membrane bag was selected to have a molecular weight of 5k and an area of 0.1m ² . Cationic chromatography packing material, the amount of sample loaded is determined according to the penetration of the small test, the pH of the sample is adjusted to pH 5.0, and the conductivity is 7.0ms/cm. Wash with pH 5.0, 0.1M NaAc buffer, then elute with 0.1M NaAc and 0.3M NaCl buffer (pH 6.0), and finally regenerate with 0.5M NaOH solution, and finally lyophilize and measure the enzyme activity, see Table 1.

2. Enzyme activity assay of lysine-specific enzymes

1) Solution preparation: Tris-HCL buffer (100 mM, pH 8.5): Weigh 2.428 g of Tris and dissolve it in 200 mg of ultrapure water, and adjust the pH to 8.5 with HCl. Lys-C substrate solution: Take a tube of 5mg substrate (N-p-Tosyl-Gly-Pro-Lys 4-nitroaniline acetate) and directly dissolve it in 3.939mL ultrapure water.

2) Experimental process: Take 180 μL of Tris-HCL buffer solution, add 10 μL of sample and 10 μL of substrate solution, mix well and use a microplate reader to read the number at 405 nm every 20 seconds for a total of 30 minutes at a reaction temperature of 30 ° C. Kinetic curve, take the linear part of the initial stage of the reaction to calculate the enzyme activity.

3) Calculation formula:

△A/t: Kinetic curve slope (take the linear part of the initial stage of the reaction)

Tv: the volume of the reaction system

Sv: volume of added sample

b: The light path is 1cm

N: Sample dilution factor

ξ: Molar extinction coefficient (9.620mL/μmol/cm for nitroaniline)

4) Definition of enzyme activity: the amount of enzyme required to catalyze the hydrolysis of 1 μmol of substrate per minute is defined as one enzyme unit.

Table 1 Enzyme activity assay results after purification and enrichment of lysine-specific enzymes

Claims (10)

1. a restructuring lysine enzyme-specific, it is characterised in that its nucleotides sequence is classified as SEQ ID NO:1.

Restructuring lysine enzyme-specific the most according to claim 1, it is characterised in that its aminoacid sequence is SEQ ID NO:2.

3. a recombinant vector, it is characterised in that described carrier is the pPIC9k-PIV containing sequence SEQ ID NO:1.

4. the genetic engineering bacterium of a high efficient expression restructuring lysine enzyme-specific, it is characterised in that described engineering bacteria is containing sequence The Pichia yeast of row SEQ ID NO:1.

Genetic engineering bacterium the most according to claim 4, it is characterised in that described genetic engineering bacterium is GS115/ pPIC9k-PIV。

6. the construction method of a high efficient expression restructuring lysine enzyme-specific engineering bacteria, it is characterised in that comprise the following steps:

1) obtain the DNA sequence SEQ ID NO:1 optimized, add 6 × His label at its C end, and at gene order 5 ' end and 3 ' End adds suitable restriction enzyme site and Kex2 site respectively, finally carries out full genome synthesis, it is thus achieved that PIV；

2) PIV that connection has leader peptide alpha factor inserts in pPIC9k, obtains recombiant plasmid pPIC9k-PIV；

3) above-mentioned gained pPIC9k-PIV is carried out Sac linearisation, and electricity goes in Pichia pastoris GS115 bacterial strain, through training Support and Enzyme activity assay, screen to obtain superior strain GS115/pPIC9k-PIV.

7. the method producing lysine enzyme-specific of recombinating described in claim 1, it is characterised in that cultivate such as claim Genetic engineering bacterium according to any one of 4～5, it is thus achieved that recombinant expressed described restructuring lysine enzyme-specific.

8. the method producing lysine enzyme-specific of recombinating described in claim 1, it is characterised in that comprise the following steps:

A) screening of lysine enzyme-specific superior strain: by inoculation to first cell culture medium, cultivate extremely at 28-30 DEG C OD₆₀₀During for 15-20, being forwarded in secondary medium, at 28-30 DEG C, concussion is cultivated, and every 8-12h adds methanol and induces Expressing, stop after fermentation 12-120h, centrifugation medium also takes supernatant；

B) fermenting and producing: by the inoculation after multiple sieve to fermentation medium, cultivates at 28-30 DEG C to dissolved oxygen amount and reaches 70- When 80%, add glycerol 9～12 hours with 2-4ml/min, add the methanol solution containing 1%～1.2%PTM1 and induce, Take fermentation liquid every 4-12h after 60-72h and carry out Enzyme activity assay；

C) by separated for above-mentioned culture fluid, obtain the protein freeze-dried powder of lysine enzyme-specific albumen after purification.

Method the most according to claim 8, it is characterised in that in step b) fermentation medium, the inoculum concentration of bacterial strain is 1- 5%.

Method the most according to claim 8, it is characterised in that in step a), pH value during methanol induction is 4.0-8.0； In step b), during methanol induction, the pH value of culture medium is 5.5-6.0.