阅读说明：本技术 一种生产重组蛋白酶k的方法 (Method for producing recombinant proteinase K ) 是由 汪本助 徐斌 王舒 于 2020-12-31 设计创作，主要内容包括：本发明涉及蛋白酶K的重组表达载体、具有所述重组表达载体的宿主细胞以及生产重组蛋白酶K的方法。本发明通过设计表达载体,实现重蛋白酶K的高效表达,再采用优化的纯化方法对目标蛋白进行提纯,大幅缩短了目标蛋白的纯化流程与纯化时间,同时提高了目标蛋白的纯度、得率和酶活,节省实验步骤并减少成本。本发明为研发与生产蛋白酶K类似的或者其他蛋白类工具酶奠定了基础。(The present invention relates to recombinant expression vectors for proteinase K, host cells having the recombinant expression vectors, and methods of producing recombinant proteinase K. According to the invention, the expression vector is designed to realize the high-efficiency expression of the heavy proteinase K, and the target protein is purified by adopting an optimized purification method, so that the purification process and the purification time of the target protein are greatly shortened, the purity, the yield and the enzyme activity of the target protein are improved, the experimental steps are saved, and the cost is reduced. The invention lays a foundation for developing the similar or other protein tool enzymes for producing the proteinase K.)

1. Recombinant expression vector for proteinase K, characterized in that a nucleotide sequence encoding proteinase K is inserted into the pPICZ α A vector plasmid.

2. The recombinant expression vector according to claim 1, wherein proteinase K is proteinase K of Candida albicans;

preferably, the nucleotide sequence encoding proteinase K is codon optimized;

preferably, the nucleotide sequence encoding proteinase K is shown in SEQ ID NO. 1.

3. The recombinant expression vector according to claim 1 or 2, wherein a nucleotide sequence encoding a protein tag, preferably a His tag, is further inserted into the recombinant expression vector.

4. A host cell having the recombinant expression vector of any one of claims 1 to 3;

preferably, the host cell is a yeast, preferably pichia pastoris.

5. A method for producing recombinant proteinase K using the recombinant expression vector of any one of claims 1 to 3 or the host cell of claim 4.

6. The method of claim 5, comprising the steps of:

(1) coating the host cell transferred with the recombinant expression vector on a bleomycin resistant plate culture medium, and culturing overnight;

(2) selecting a single colony, culturing the single colony in a bleomycin resistant liquid culture medium, and adding an inducer to induce protein expression to obtain a fermentation liquor;

(3) and filtering the fermentation liquor, removing thalli, collecting supernatant, and purifying.

7. The method according to claim 6, wherein in the step (2), the pH value of ammonia water is controlled to be 4.5-5.5 during fermentation, and dissolved oxygen is maintained to be between 20% and 35%;

and/or during fermentation, when the dissolved oxygen rises back to more than 80%, the glycerol culture medium is supplemented, and when the bacterial concentration reaches 180-;

and/or when the dissolved oxygen rises to 100% in the fermentation period, after starving for half an hour, beginning to supplement the methanol induction culture medium, and after the induction expression is carried out for 40-55 hours, putting the culture medium into a tank.

8. The method according to claim 7, wherein the glycerol medium comprises 45-55% of glycerol by mass and 3.5-5 ml/L of trace element PTM 1;

and/or the methanol induction culture medium is a methanol solution containing 3.5-5 ml/L trace element PTM 1.

9. The method of claim 6, wherein the filtering comprises: firstly, filtering by using a filter membrane with the aperture smaller than 1 micron, and then filtering by using a filter membrane with the aperture smaller than 25 KDa.

10. The method according to claim 6, wherein the purification comprises passing the supernatant through an affinity chromatography column, preferably a nickel ion metal chelate affinity chromatography column, Ni²⁺-NTA；

Preferably, the purification comprises: and adding the supernatant into the affinity chromatography column, washing with a buffer solution containing 70-90 mM imidazole to remove foreign proteins, and then eluting with a buffer solution containing 400-600 mM imidazole to obtain a solution containing the target protein.

Technical Field

The invention relates to the field of molecular biology, in particular to a method for producing recombinant proteinase K.

Background

Proteinase K (EC 3.4.21.14) is derived from Candida albicans (Tritirachium album Limber), a subtilisin-related serine protease, and was named proteinase K because it degrades keratin (keratin). Proteinase K consists of a peptide chain containing 277 amino acid residues, has the relative molecular mass of 28930, consists of an active site D39, H69 and S224, and mainly comprises two fragments of G100-Y104 and S132-G136. Proteinase K contains two disulfide bonds (C34-C124, C179-C248), two tryptophan residues (W8, W212) and one free cysteine (C73). X-ray crystallography studies have shown that the three-dimensional structure of proteinase K is a well-defined spherical fold. Proteinase K belongs to the alpha/beta class of proteins, comprising 6 alpha-helices, 15 beta-sheets and an 3/10 helix.

Proteinase K is a high-activity broad-spectrum proteinase, has higher proteolytic activity to both natural protein and non-natural protein, and still has higher activity in the environment containing urea or Sodium Dodecyl Sulfate (SDS), and is widely applied in the aspects of biological experiments, paint washing industry, sewage treatment and the like due to the important property of proteinase.

At present, the common PK production method is mainly extracted from PK-producing microorganisms, and has the defects of complicated steps and low protein acquisition amount. Therefore, it is very necessary to develop a method for producing recombinant PK in pichia pastoris, so as to realize the preparation of high-purity recombinant PK, thereby saving experimental steps and reducing cost.

Disclosure of Invention

The present invention aims to overcome the defects of the prior art and provide a recombinant expression vector of proteinase K, a host cell with the recombinant expression vector and a method for producing the recombinant proteinase K.

In a first aspect, the present invention provides a recombinant expression vector of proteinase K, wherein a nucleotide sequence encoding proteinase K is inserted into a pPICZ α a vector plasmid. The invention can use pPICZ alpha A-PK to represent the recombinant expression vector.

According to the invention, a large number of practices show that the recombinant expression vector of the proteinase K obtained by utilizing pPICZ alpha A vector plasmid can realize the high-efficiency expression of the proteinase K.

In some embodiments, the proteinase K is proteinase K of candida albicans.

In some embodiments, the nucleotide sequence encoding proteinase K is codon optimized for expression in a host cell.

In some embodiments, the nucleotide sequence encoding proteinase K is shown in SEQ ID NO:1, specifically:

GCCCCAGCTGTTGAACAAAGATCTGAAGCTGCTCCATTGATTGAAGCTAGAGGTGAAATGGTTGCTAACAAGTACATTGTTAAGTTTAAGGAAGGTTCAGCTTTGTCTGCTTTGGATGCTGCTATGGAAAAGATTTCTGGTAAACCAGATCATGTTTACAAGAACGTGTTTTCAGGTTTTGCTGCTACTTTGGATGAAAATATGGTTAGAGTTTTGAGAGCTCATCCTGATGTTGAATATATTGAACAAGATGCTGTTGTTACTATTAACGCAGCTCAAACTAACGCTCCTTGGGGTTTGGCTAGAATTTCTTCTACTTCTCCAGGTACTTCTACTTACTATTACGATGAATCTGCTGGTCAAGGATCTTGTGTTTACGTTATTGATACTGGAATTGAAGCTTCTCATCCAGAATTTGAAGGTAGAGCTCAAATGGTTAAGACTTACTACTACTCTTCTAGAGATGGTAATGGACATGGAACTCATTGTGCTGGTACTGTTGGATCTAGAACTTACGGAGTTGCTAAGAAAACACAATTGTTCGGTGTTAAGGTTTTGGATGATAACGGTTCTGGTCAATACTCAACTATTATTGCTGGTATGGATTTTGTTGCTTCTGACAAGAATAACAGAAATTGTCCTAAAGGAGTTGTTGCTTCTTTGTCTTTGGGTGGTGGTTACTCTTCATCAGTTAATTCTGCTGCTGCTAGATTGCAATCATCCGGTGTTATGGTTGCTGTTGCTGCTGGTAATAATAATGCTGATGCTAGAAATTACTCTCCAGCTTCAGAACCATCTGTTTGTACTGTTGGTGCTTCTGATAGATATGATAGAAGATCTTCTTTCTCTAACTACGGTTCTGTTTTGGATATTTTCGGTCCTGGTACTTCTATTTTGTCCACCTGGATTGGTGGTTCTACTAGATCTATTTCTGGTACTTCTATGGCTACTCCACATGTTGCTGGTTTAGCTGCCTATCTGATGACTTTGGGAAAAACTACTGCCGCTTCTGCTTGTAGATACATTGCTGATACTGCTAATAAGGGTGACTTGTCCAACATTCCTTTTGGTACTGTTAACTTGTTGGCTTACAACAATTACCAAGCTTAA

in some embodiments, the recombinant expression vector further has inserted therein a nucleotide sequence encoding a protein tag. Preferably, the protein tag is a His tag in the invention, so that protein purification can be conveniently carried out in the production process.

In some embodiments, the recombinant expression vector is prepared by a method comprising: synthesizing the proteinase K gene according to the nucleotide sequence after codon optimization; the pPICZ alpha A vector plasmid is linearized through enzyme digestion, and the linearized product and the synthetic proteinase K gene are seamlessly constructed to form a recombinant expression vector.

The invention also requires verification of the recombinant expression vector to facilitate screening for positive clones. The verification may be performed using methods known in the art.

In some embodiments, the verifying comprises the steps of: adding 10ul of recombinant product into DH5 alpha competent cells, standing on ice for 30min, then thermally shocking at 42 ℃ for 90s, standing on ice for 3min, adding 400ul of LB liquid culture medium, shake culturing at 37 ℃ and 200rpm for 1h, coating the mixture on an LLB plate containing 25ug/mL bleomycin, and finally putting the LLB plate into a 37 ℃ incubator for overnight culture; d. randomly picking four colonies from an LB plate by using a sterilized toothpick, and inoculating the four colonies into 4ml of bleomycin-resistant LLB culture medium; shaking culture was carried out overnight at 37 ℃ and 180rpm, and plasmids were extracted and sequenced. The sequencing may be performed by sequencing methods known in the art, such as Sanger's method, and the like.

In a second aspect, the present invention provides a host cell, preferably a yeast, having the recombinant expression vector.

In some embodiments, the host cell is pichia pastoris.

The method provided by the invention can realize the high-efficiency expression of the pPICZ alpha A-PK recombinant expression vector in pichia pastoris.

In a third aspect, the invention provides a method for producing recombinant proteinase K using said recombinant expression vector or said host cell.

In some embodiments, the method comprises the steps of:

(1) coating the host cell transferred with the recombinant expression vector on a bleomycin resistant plate culture medium, and culturing overnight;

(2) selecting a single colony, culturing the single colony in a bleomycin resistant liquid culture medium, and adding an inducer to induce protein expression to obtain a fermentation liquor;

(3) and filtering the fermentation liquor, removing thalli, collecting supernatant, and purifying.

In some embodiments, the culturing of step (1) is performed overnight at 30 ℃ and the plate dish is inverted.

In some embodiments, the culturing of the single colony in the liquid medium in step (2) is specifically: and scraping the bacterial colonies, inoculating the bacterial colonies into an YPD liquid culture medium, and directly inoculating the liquid culture medium into a yeast fermentation culture medium for culture when the OD600 reaches 4.0-8.0.

In some embodiments, the pH value of the ammonia water during the fermentation in the step (2) is controlled to be 4.5-5.5, and the dissolved oxygen is maintained between 20% and 35%.

In some embodiments, the glycerol medium is started to be supplemented when the dissolved oxygen rises back to more than 80% during the fermentation in the step (2), and the glycerol medium is stopped to be supplemented when the bacterial concentration reaches 180-220 g/L.

In some embodiments, the glycerol medium comprises 45-55% of glycerol by mass and 3.5-5 ml/L of trace element PTM 1.

In some embodiments, when the dissolved oxygen rises back to 100% during the fermentation in step (2), after starvation for half an hour, the methanol induction medium starts to be supplemented, and after the induction expression is carried out for 40-55 hours, the tank is placed.

In some embodiments, the methanol induction medium is a methanol solution containing 3.5-5 ml/L trace element PTM 1.

The method provided by the invention can also be used for detecting thalli before and after induction expression so as to ensure successful induction. The detection may be by methods known in the art.

In some embodiments, the detecting comprises the steps of: picking a single colony on a plate, placing the single colony in a YPD culture medium containing bleomycin resistance, culturing overnight at 30 ℃ and 200rpm, inoculating bacteria in 100ml of BMGY culture medium containing bleomycin according to the volume ratio of 1:100, continuously culturing for 24h, centrifugally collecting bacteria, transferring the bacteria into a BMMY culture medium, and culturing at 30 ℃ for 120h to induce protein expression; respectively taking bacterial liquid before and after induction expression, respectively centrifuging at 8000rpm for 20min, collecting supernatant, adding 5 Xprotein electrophoresis buffer, boiling for 5min, cooling, centrifuging at 12000rpm for 5min, taking 20ul of processed sample, performing 15% polyacrylamide gel electrophoresis, taking down gel, dyeing and decolorizing for 10min by using a rapid dyeing instrument, and observing the change of protein bands before and after induction.

In some embodiments, the filtering of step (3) comprises: filtering with a filter membrane (such as ceramic membrane) with pore diameter smaller than 1 μm, and filtering with a filter membrane (such as ultrafiltration membrane) with pore diameter smaller than 25 KDa.

In some embodiments, the purifying of step (3) comprises passing the supernatant through an affinity chromatography column, preferably a nickel ion metal chelate affinity chromatography column, Ni²⁺-NTA。

In some embodiments, the affinity chromatography column is washed repeatedly before use, and then the affinity chromatography column is equilibrated with a buffer.

In some embodiments, the purifying comprises: and adding the supernatant into the affinity chromatography column, washing with a buffer solution containing 70-90 mM imidazole to remove foreign proteins, and then eluting with a buffer solution containing 400-600 mM imidazole to obtain a solution containing the target protein.

In some embodiments, the buffer used for purification is a NAT buffer, specifically consisting of: 20mM Tris-HCl pH7.9, 0.5M NaCl.

According to the invention, the optimized method is adopted to purify the proteinase K, the whole purification process is simple and convenient to operate, the purification process and the purification time of the proteinase K are greatly shortened, the purity, the yield and the enzyme activity of the proteinase K are simultaneously improved, the proteinase K fused with the His tag can carry out enzyme digestion and purification on the target protein with the His tag, the effluent liquid of the Ni column after enzyme digestion is collected to obtain the natural protein with higher concentration and purity, and compared with other enzyme digestion and purification modes, the experimental steps are saved and the cost is reduced.

The purified protein solution can be detected by SDS-PAGE.

Drawings

FIG. 1 is a graph comparing the results of electrophoresis before and after induction of recombinant proteinase K; wherein lane 1 represents the supernatant before induction; lane 2 represents the supernatant after induction.

FIG. 2 is a graph comparing results before and after concentration by ultrafiltration of proteinase K; wherein lane 1 represents the sample before ultrafiltration; lane 2 represents the sample after ultrafiltration.

FIG. 3 shows the result of electrophoresis detection after protease K affinity chromatography purification.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1: construction of pPICZ alpha A-PK recombinant vector

According to the gene sequence of the Linburg candida albicans proteinase K gene after codon optimization, directly carrying out gene synthesis on proteinase K; the pPICZ alpha A vector plasmid is linearized through enzyme digestion, and the linearized product and a gene synthesis product are seamlessly constructed to form a recombinant product.

Transferring 20ul of recombinant product into DH5 alpha competent cells, standing on ice for 30min, then thermally shocking at 42 ℃ for 90s, standing on ice for 2min, then adding 400ul of LLB liquid culture medium, performing shake culture at 37 ℃ and 200rpm for 1h, coating the mixture on a LLB plate containing bleomycin, and finally putting the LLB plate into a 37 ℃ incubator for overnight culture; randomly picking four colonies from the LLB plate by using a sterilized toothpick, and inoculating the four colonies into 4ml of the bleomycin-resistant LLB culture medium; the cells were subjected to shake culture at 37 ℃ and 200rpm overnight, and the plasmids were extracted and sequenced by Sanger's method.

Example 2 inducible expression and characterization of proteinase K

The pPICZ alpha A-PK plasmid which is identified as positive by a Sanger method sequencing result is converted into competent Pichia pastoris after enzyme digestion linearization, coated on a YPD plate containing bleomycin resistance, and inversely cultured for 72h at 30 ℃.

Picking a single colony on the YPD plate by using a sterilized toothpick, placing the single colony in a YPD culture medium containing bleomycin resistance, culturing overnight at the temperature of 30 ℃ and the speed of 200rpm, inoculating bacteria in a BMGY culture medium according to the volume ratio of 1:100, and continuously culturing for 24 hours; centrifuging BMGY cultured bacteria liquid to collect thalli, removing supernatant, transferring the thalli into an isometric BMMY culture medium, and carrying out induction culture for 120 h.

Taking the induced expression bacterial liquid, centrifuging for 20min at 8000rpm, collecting supernatant, adding 5 Xprotein electrophoresis buffer, boiling for 5min, cooling, centrifuging for 5min at 12000rpm, taking 20ul electrophoresis buffer sample, performing 15% polyacrylamide gel electrophoresis, taking down the gel, dyeing and decolorizing for 10min by using a rapid dyeing instrument, and observing the change of protein bands before and after induction.

The results are shown in FIG. 1. The results showed that the cell after induction expression showed an expression band corresponding to the theoretical molecular weight of PK, but the cell before induction did not show the expression band, thus confirming that PK expression was normal.

Example 3: proteinase K fermentation

And (3) coating the positive PK pichia pastoris bacterial liquid on a YPD plate, culturing the YPD plate for 20 hours at the temperature of 30 ℃, scraping bacterial colonies, inoculating the bacterial colonies into a YPD liquid culture medium, and directly inoculating the liquid culture medium into a yeast fermentation culture medium when the OD600 reaches 6.0. During fermentation, the pH of ammonia water is controlled to be 5.0, and dissolved oxygen is maintained to be between 20 and 35 percent. And (3) beginning to supplement the glycerol culture medium when the fermentation dissolved oxygen rises back to more than 80%, and stopping supplementing the glycerol culture medium when the bacterial concentration reaches 180-220 g/L. And when the dissolved oxygen rises back to 100%, starving for half an hour, then beginning to supplement the methanol induction culture medium, and putting the culture medium into a tank after 48 hours of induction.

Wherein, the glycerol culture medium is 50 percent (mass percentage) of glycerol, and PTMI4.35ml/L is added; the methanol induction medium is 100 percent of methanol, and PTMI4.35ml/L is added.

Example 4: proteinase K purification

Filtering the fermentation liquor obtained in the fermentation in the embodiment 3 by using a ceramic membrane (the aperture is less than 1 micron) to remove thalli, and collecting the supernatant; the supernatant collected by the ceramic membrane filtration was concentrated using an ultrafiltration membrane (pore size less than 25kDa) to remove a part of the solution (SDS-PAGE results of the sample before and after concentration are shown in FIG. 2).

The column was washed with 5 column volumes of pure water, and the washing was repeated twice, followed by equilibration of the column with 5 column volumes of NAT-0 buffer (20mM Tris-HCl pH7.9, 0.5M NaCl).

Adding supernatant obtained by concentrating ultrafiltration membrane to Ni²⁺-NTA chromatography column, after repeated addition 2-3 times, washing the medium with 5 column volumes of NAT-1 buffer (i.e. NAT-0 buffer containing 80mmol/L imidazole) to remove contaminating proteins, and finally eluting the desired protein with the above buffer containing 300mmol/L imidazole to obtain purified PK.

The SDS-PAGE results of the purified protein solutions are shown in FIG. 3. From the results of this figure, it can be seen that the protein of interest is enriched in solution.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> Anhui Feng Yuan fermentation engineering research Co Ltd

<120> a method for producing recombinant proteinase K

<130> RYP2011024.0

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1110

<212> DNA

<213> Artificial Sequence

<220>

<223> proteinase K

<400> 1

gccccagctg ttgaacaaag atctgaagct gctccattga ttgaagctag aggtgaaatg 60

gttgctaaca agtacattgt taagtttaag gaaggttcag ctttgtctgc tttggatgct 120

gctatggaaa agatttctgg taaaccagat catgtttaca agaacgtgtt ttcaggtttt 180

gctgctactt tggatgaaaa tatggttaga gttttgagag ctcatcctga tgttgaatat 240

attgaacaag atgctgttgt tactattaac gcagctcaaa ctaacgctcc ttggggtttg 300

gctagaattt cttctacttc tccaggtact tctacttact attacgatga atctgctggt 360

caaggatctt gtgtttacgt tattgatact ggaattgaag cttctcatcc agaatttgaa 420

ggtagagctc aaatggttaa gacttactac tactcttcta gagatggtaa tggacatgga 480

actcattgtg ctggtactgt tggatctaga acttacggag ttgctaagaa aacacaattg 540

ttcggtgtta aggttttgga tgataacggt tctggtcaat actcaactat tattgctggt 600

atggattttg ttgcttctga caagaataac agaaattgtc ctaaaggagt tgttgcttct 660

ttgtctttgg gtggtggtta ctcttcatca gttaattctg ctgctgctag attgcaatca 720

tccggtgtta tggttgctgt tgctgctggt aataataatg ctgatgctag aaattactct 780

ccagcttcag aaccatctgt ttgtactgtt ggtgcttctg atagatatga tagaagatct 840

tctttctcta actacggttc tgttttggat attttcggtc ctggtacttc tattttgtcc 900

acctggattg gtggttctac tagatctatt tctggtactt ctatggctac tccacatgtt 960

gctggtttag ctgcctatct gatgactttg ggaaaaacta ctgccgcttc tgcttgtaga 1020

tacattgctg atactgctaa taagggtgac ttgtccaaca ttccttttgg tactgttaac 1080

ttgttggctt acaacaatta ccaagcttaa 1110