CN102839165A - Gene mutation type recombined protease K and industrialized production method thereof - Google Patents
Gene mutation type recombined protease K and industrialized production method thereof Download PDFInfo
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- CN102839165A CN102839165A CN2012103637008A CN201210363700A CN102839165A CN 102839165 A CN102839165 A CN 102839165A CN 2012103637008 A CN2012103637008 A CN 2012103637008A CN 201210363700 A CN201210363700 A CN 201210363700A CN 102839165 A CN102839165 A CN 102839165A
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Abstract
The invention relates to a gene mutation type recombined protease K and an industrialized production method thereof. Specifically, the invention relates to the gene mutation type recombined protease K which is obtained by performing gene reformation and protein engineering and has an enzymatic activity being more than two times of the enzymatic activity of the same natural protein, and further relates to the industrialized production method and a technical process for utilizing yeast cells to large-scale culture expressing foreign proteins and prepare the gene mutation type recombined protease K.
Description
Technical field
The present invention relates to a kind of genic mutation type recombinant protein enzyme K and industrialized preparing process thereof.More specifically, the present invention relates to a kind of enzymic activity that obtains through genetic modification and protein engineeringization is the genic mutation type recombinant protein enzyme K of wild-type protease activity of the same race more than 2 times; And utilize the yeast cell large scale culturing to express foreign protein, prepare industrialized preparing process and the technological process of said genic mutation type recombinant protein enzyme K.
Background technology
Proteinase K was a kind of Tryase, in the extract of woods Bai Shi Candida albicans (Tritirachium album limber), found (Ebeling W etc., Eur J Biochem.1974 Aug 15 first in 1974; 47 (1): 91-7).Because the natural keratin of disulfide linkage is rich in this endonuclease capable digestion, woods Bai Shi Candida albicans can be with Keratin sulfate as unique carbon source/nitrogenous source growth, and Proteinase K is also gained the name thus.
The maximum characteristics of Proteinase K are natural protein is had the cutting power of broad-spectrum high efficacy, are active the highest a kind of in the existing proteolytic enzyme.This enzyme prefers to the carboxy-terminal peptide bond of cutting aliphatic amino acid and die aromatischen Aminosaeuren, particularly L-Ala.
In addition; Proteinase K still is the very outstanding enzyme of a kind of stability; Under the condition of 12,20 ℃ ~ 60 ℃ of pH 4 ~ pH, activity is arranged all, in the multiple chemical reagent (like SDS, urea, sequestrant (like EDTA) and SR (like trypsin inhibitor or chymotrypsin inhibitor)) that can make protein denaturation, also have the ability of scinderin matter.
Owing to use SDS solution in the lysis process usually; And Proteinase K still can be kept activity completely in the SDS solution of 0.5% (w/v); Make it can be applicable in the nucleic acid extraction, nucleicacidase in the born of the same parents that discharge through quick degradation of cell cracking separates obtaining complete nucleic acid fragment.Research formerly shows that also Proteinase K also has outstanding stability in stain remover (like Triton X-100), thereby also is widely used in the research of membranin and transmembrane protein.
In recent years, the application of Proteinase K not merely is confined to medical diagnosis and scientific research field, in fields such as industrial process hides, papermaking, feed, also can substitute traditional chemical process that possibly have pollution and handle starting material.Therefore, the Proteinase K that obtains high reactivity, low cost has practical significance.
Yet neutral protease K relies on traditional zymotic to extract, and output is very low, and cost is higher, is not suitable for the extensive utilization of this product from now on.Therefore, the research focus of this area concentrates on and utilizes genetic engineering technique and protein engineering at present, the molecular structure of engineered protein, and expectation obtains the Proteinase K that activity is higher, cost is lower.
Up to now, the gene recombinant protein enzyme K of existing suitability for industrialized production is to be the host with the prokaryotic cell prokaryocyte, such as escherichia expression system, and its advantage is easy, economical.Yet the Proteinase K activity that this system expression obtains is not high or do not have activity, does not reach the level of neutral protease K, and this possibly be that prokaryotic system can't be realized this function because last handling process need transcribed or translate to Proteinase K.In addition, it is low excessively to utilize prokaryotic cell prokaryocyte to carry out the output of exogenous protein expression, thereby is difficult to meet the need of market.
In addition, if seek out commercial Proteinase K product, downstream purification and freeze drying technology are extremely important, the data that can not use for reference in this respect at present.For example, utilize that the purification process reported all can not obtain in enormous quantities, the Proteinase K of high yield.Through the inventor's test, these purification process practical value in large-scale production is not high.
For addressing the above problem, the inventor has carried out deep research, in conjunction with transgenation and eukaryotic cell-yeast external secretion expression technology, starts with from engineered protein enzyme K molecular structure, obtains the active genic mutation type recombinant protein enzyme K that improves; The optimization expression system utilizes yeast cell external secretion expression technology, and large scale fermentation efficiently expresses genic mutation type recombinant protein enzyme K, further improves the activity and the output of Proteinase K; Adopt novel protein purifying mode, improve the yield in the Proteinase K purge process, thereby realized mass-producing and serialization production, accomplished the present invention.In patent document and non-patent document, all do not find at present report about genic mutation type recombinant protein enzyme K suitability for industrialized production in yeast.
Summary of the invention
Wild-type protease K precursor is formed (SEQ ID NO.1 by 384 amino acid; UniProtKB/Swiss-Prot:P06873.2), comprise the propetide of the signal peptide of 15 amino acid lengths, 90 amino acid lengths and the maturation protein enzyme K of 279 amino acid lengths.
Through external scientist's research, than wild-type protease K, genic mutation type recombinant protein enzyme K can have higher activity.The inventor is from aminoacid sequence (the SEQ ID NO.2 of wild-type protease K; Form by 369 amino acid; Comprise the propetide of 90 amino acid lengths and the maturation protein enzyme K of 279 amino acid lengths) in selected eight critical sites; Synthetic gene is also expressed successfully at eukaryotic system, has obtained the genic mutation type recombinant protein enzyme K that enzyme is lived and improved greatly.
Therefore, first aspect of the present invention is to provide a kind of genic mutation type recombinant protein enzyme K, it is characterized in that:
(1) the 151st Y in the wild-type protease K aminoacid sequence (SEQ ID NO.2) being sported A, the 208th K sports H, the 273rd S and sports T, the 293rd G and sport that A, the 332nd K sport R, the 337th S sports N; Randomly, also carry out following any sudden change more than a kind:
(2) the 123rd S is sported A;
(3) the 180th L is sported I.
The present invention is to provide the DNA of coding said gene mutant recombinant protein enzyme K on the other hand.
The present invention is to provide the expression vector that comprises above-mentioned DNA on the other hand.
Another aspect of the invention is to provide the transformant that imports above-mentioned expression vector.
Further aspect of the present invention is to provide the test kit that contains said gene mutant recombinant protein enzyme K, DNA, expression vector and/or transformant.
The present invention is to provide the method for utilizing eukaryotic cell to prepare said gene mutant recombinant protein enzyme K on the other hand, and this method comprises following steps: in substratum, cultivate the transformant that contains genic mutation type recombinant protein enzyme K of the present invention; And collect the said genic mutation type recombinant protein enzyme K in the said substratum, it is characterized in that said transformant is a yeast cell.
In addition, the inventor has obtained commercial genic mutation type recombinant protein enzyme K product through selecting the decolouring of different resins material, different layers chromatography medium purification, different protective material and freeze-dry process lyophilize.
Therefore, after the present invention provides fermentation production of protein enzyme K on the other hand, adopt novel purifying mode to handle and improve yield and the cryodesiccated technology of batch.
Description of drawings
From with the following detailed description of accompanying drawing bonded, will more be expressly understood above and other objects of the present invention, characteristic and other advantage, wherein:
Fig. 1 representes to show the synoptic diagram in mutational site in genic mutation type recombinant protein enzyme K mutProK6 of the present invention, mutProK7-1, mutProK7-2 and the mutProK8 aminoacid sequence.
Fig. 2 representes the carrier collection of illustrative plates of Yeast expression carrier pPIC9K.
Fig. 3 representes to utilize SDS-PAGE to detect result's (laboratory scale of the expression of genic mutation type recombinant protein enzyme K mutProK8 of the present invention in the GS115 yeast cell; Shake flask fermentation).
Fig. 4 representes to utilize the Western trace to detect result's (laboratory scale of the expression of genic mutation type recombinant protein enzyme K mutProK8 of the present invention in the GS115 yeast cell; Shake flask fermentation and 30L ferment tank).
Fig. 5 representes result's (laboratory scale of the native polyacrylamide gel electrophoresis of genic mutation type Proteinase K mutProK8 and wild-type protease K; Shake flask fermentation).
Fig. 6 representes the qualitative experiment result of genic mutation type Proteinase K mutProK8 digestion bovine serum albumin (BSA).
Resulting tyrosine typical curve when Fig. 7 representes to utilize the F-C analytical method that the enzymic activity of genic mutation type recombinant protein enzyme K of the present invention is carried out qualitative detection.
Fig. 8 representes to utilize SDS-PAGE to detect the result of 2 tons of fermentor cultivation pichia spp different time yield of enzyme.
The result that the purity of genic mutation type recombinant protein enzyme K after Fig. 9 representes to utilize electrophoresis to purified freeze-drying detects: Fig. 9 A: denaturing polyacrylamide gel electrophoresis; Fig. 9 B: native polyacrylamide gel electrophoresis.
Figure 10 representes the stability result of genic mutation type recombinant protein enzyme K of the present invention.
Embodiment
Hereinafter will be set forth the present invention in detail.
One. genic mutation type recombinant protein enzyme K of the present invention and determination of activity
Genic mutation type recombinant protein enzyme K of the present invention is characterized in that:
(1) the 151st Y in the wild-type protease K aminoacid sequence (SEQ ID NO.2) being sported A, the 208th K sports H, the 273rd S and sports T, the 293rd G and sport that A, the 332nd K sport R, the 337th S sports N; Randomly also carry out following any sudden change more than a kind:
(2) the 123rd S is sported A;
(3) the 180th L is sported I.
In the present invention; It is not the absolute location that must represent from the N end of said Proteinase K that " 123 ", " 151 ", " 180 ", " 208 ", " 273 ", " 293 ", " 332 " reach " 337 ", but the relative position that expression is compared with the aminoacid sequence of SEQ ID NO.2.For example, in the proteolytic enzyme of the aminoacid sequence that contains SEQ ID NO.2, when the N of 123 in this proteolytic enzyme amino acid of having held a certain topagnosis, above-mentioned 123 promptly become 122.Even under this situation, said is that 122 amino acid still is the amino acid of " 123 " in the present invention from N end residue counting.Through being compared, the aminoacid sequence of the aminoacid sequence of protein of interest enzyme and SEQ ID NO.2 confirms said amino acid whose relative position.
Compare with the enzymic activity of corresponding wild-type proteolytic enzyme (SEQ IDNO.2), the enzymic activity of genic mutation type recombinant protein enzyme K of the present invention is higher.
According to the present invention one preferred embodiment, can measure the activity of recombinant protein enzyme of the present invention through following method.
Casein food grade is dissolved in the potassium phosphate solution and regulates pH value preparation substrate solution.Sample is added in the above-mentioned substrate solution.Make this mixture 37 ℃ of reactions down, add the reaction terminating liquid termination reaction then.Through the centrifugal supernatant that obtains, add soda ash light, add phenol reagent (Folin-Ciocalteu reagent) then.Absorbancy is measured in the reaction back under 660nm.To deduct the content that value that the barren absorbance obtains is converted into free tyrosine with the absorbance of sample, to calculate the protein-active value.The unit of protein-active is U/mL (units per ml).This 1 unit is meant in aforesaid method, the amount of the enzyme that the feasible phenol reagent colour developing thing that is equivalent to 1 μ mol tyrosine increases in 1 minute (min).Obtain the tie-in equation of tyrosine and phenol reagent colour developing thing through the working curve of making tyrosine.
Two. the DNA of the genic mutation type recombinant protein enzyme K of the present invention that encodes
DNA of the present invention is the DNA of coding genic mutation type recombinant protein enzyme K of the present invention.One preferred embodiment according to the present invention; DNA of the present invention can obtain in the following manner: for example adopt method such as PCR (for example to obtain wild-type protease K gene; Nucleotide sequence shown in SEQ ID NO.3); The purpose sudden change is imported the DNA of each genic mutation type recombinant protein enzyme K of preparation coding through fixed-point mutation method etc.
Do not limit for fixed-point mutation method is special, for example, can use commercially available QuikChange Site-Directed Mutagenesis Kit (Stratagene manufactured) etc. to carry out.As the method for introducing rite-directed mutagenesis, for example, Gapped duplex method and Kunkel method all are known.
Another preferred embodiment also can obtain DNA of the present invention through chemosynthesis according to the present invention.According to a particularly preferred embodiment of the present; For improving the expression efficiency of recombinant protein in host cell; Can the encoding sequence (SEQ ID NO.3) of wild-type protease K be replaced with the dna sequence dna that the host cell preference codon is formed; After introducing the purpose sudden change, the mode through chemosynthesis prepares DNA of the present invention again.Most preferred embodiment according to the present invention, said host cell preference codon is the yeast preference codon.
Preferably, as the DNA of coding genic mutation type recombinant protein enzyme K of the present invention, for example be DNA with nucleotide sequence of the represented 7-1113 position of SEQ ID NO.6-9.But, as long as coding genic mutation type recombinant protein enzyme K of the present invention then is not limited to these.
In addition; The encode polynucleotide of genic mutation type recombinant protein enzyme K of the present invention also can be the polynucleotide of the following genic mutation type recombinant protein enzyme K of coding: under stringent condition, hybridize and encode with the complementary sequence of the nucleotide sequence of the represented 7-1113 position of SEQ ID NO.6-9 and have above-mentioned specific sudden change and have above-mentioned desirable active genic mutation type recombinant protein enzyme K.
Stringent condition is meant the condition that forms so-called specific hybrid and do not form non-specific hybridization.Although this condition is because of nucleotide sequence or its length difference; The example comprises (for example having high homology; Have and be not less than 75%, preferably be not less than 90%, further preferably be not less than 95% homology) the mutual cross of DNA phase, and homology is lower than the condition that the DNA of above-mentioned standard is not hybridized; Or be used in the Southern hybridization typical conditions of rinsing hybridization conditions (60 ℃ with 1 * SSC, 0.1%SDS, preferred 0.1 * SSC and be equivalent to the salt concn of 0.1%SDS).
Three. expression vector of the present invention
Expression vector of the present invention is the expression vector that is used to express genic mutation type recombinant protein enzyme K of the present invention.According to the present invention one preferred embodiment, said expression vector can have following structure: control the upper reaches of DNA that said DNA expression promoter sequence is connected to coding genic mutation type recombinant protein enzyme K of the present invention.In addition, can also terminator be connected to the downstream of said DNA.
As the expression vector in the yeast cell, can use any kind among pPICZ, pPICZ α, pGAPZ, pGAPZ α, pGAPZ α A and the pPIC9K.From the angle of copy number with stability, preferred pPIC9K.
According to the present invention one preferred embodiment, be used for selecting the selectable marker gene of recombinant chou or be used to detect the reporter gene that quiding gene expresses also can inserting expression vector of the present invention.The instance of selectable marker gene includes but not limited to hygromycin gene, kalamycin resistance gene and ampicillin resistance gene.The instance of reporter gene includes but not limited to beta-Glucuronidase (GUS) gene, E.C. 2.3.1.28 (CAT) gene, luciferase (LUC) gene and green fluorescent protein (GFP) gene.
Another preferred embodiment for secreting, expressing genic mutation type recombinant protein of the present invention enzyme K or for the ease of the expressed Proteinase K of purifying, can comprise appended sequence in the expression vector of the present invention according to the present invention.In this case, Proteinase K of the present invention is with the formal representation of fusion rotein (merging with appended sequence encoded protein or peptide).The instance of said appended sequence includes but not limited to the nucleotide sequence of coded signal peptide or propetide; And the nucleotide sequence of coding His label or GST label.
Four. transformant of the present invention
Transformant of the present invention is the cell that imports expression vector of the present invention, and this cell can be produced genic mutation type recombinant protein enzyme K of the present invention.Although this transformant can be a prokaryotic cell prokaryocyte can be eukaryotic cell maybe, be preferably eukaryotic cell.
Said eukaryotic instance includes but not limited to yeast cell.According to the present invention one preferred embodiment, said eukaryotic cell is a yeast cell.According to the present invention another preferred embodiment, said yeast cell is pichia spp (Pichia) cell, candiyeast (Candida) cell, many types of debaryomyces hansenii (Hansenula polymorpha) cell, torulopsis (Torulopsis) cell, fission yeast (Schizosaccharomyces) cell and yeast kluyveromyces fragilis (Kluyveromyces) cell.
Especially preferred embodiment according to the present invention, said yeast cell is the pichia spp cell.Because the fermentation condition to pichia spp has more deep research at present; And said pichia spp cell cost in fermentative prepn is minimum; Can satisfy the demand of technical need and product marketization, thereby the pichia spp cell is considered to highly preferred transformant in the genic mutation type recombinant protein enzyme K production.
According to the present invention one preferred embodiment, said pichia spp cell is GS115 yeast cell (U.S. Gproan Protein Engineering, Inc. gives).
Can suitably select expression vector is imported the method for transformant according to the type of transformant.These methods all are well known by persons skilled in the art.
According to the present invention one preferred embodiment, can obtain the transformant of pichia spp through following method: the method that adopts electric shock to transform is transformed into the linearizing expression vector in the yeast competent cell.Subsequently the thalline suspension is coated on the flat board, and culture plate is until single bacterium colony occurring.
Five. test kit of the present invention
Test kit of the present invention is one or more the test kit that comprises in DNA, expression vector of the present invention and the transformant of the present invention of genic mutation type recombinant protein enzyme K of the present invention, coding genic mutation type recombinant protein enzyme K of the present invention.
Usually, said test kit comprises container and on this container or the label or the package insert that are associated with this container.Suitable containers comprises, for example, and bottle, bottle, syringe etc.This container can be formed by multiple material, like glass or plastics.Said label and package insert have been indicated the method for use and the purposes of this test kit.
Randomly; Test kit of the present invention also can comprise one or more components in addition; Said component is selected from the group that is made up of test tube, reaction buffer, PCR primer, dNTP, Taq polysaccharase, reversed transcriptive enzyme, DNA enzyme, RNA enzyme inhibitors, DEPC water and sterilized water, but always is not limited to this.
Six. preparing method's (laboratory scale) of genic mutation type recombinant protein enzyme K of the present invention
Through cultivating transformant of the present invention; Can produce genic mutation type recombinant protein enzyme K of the present invention; Formal representation genic mutation type recombinant protein of the present invention enzyme K through the fusion rotein that merges with signal peptide is to be used for secretion, and proteolytic enzyme of the present invention can accumulate in substratum.When using inducible promoter, preferably between during cultivation, induce.Although it is different with the type of cell to cultivate the method for said transformant, can use traditional method.
According to the present invention one preferred embodiment, the instance of cultivating the method for said pichia spp transformant is described below:
Cultivate in picking yeast list bacterium colony to the YPD substratum (yeast extract 10g/L, peptone 20g/L, glucose 20g/L).Centrifugal collection thalline.With MM substratum (13.4g/L YNB, 4 * 10
-4G/L vitamin H, 5mL/L methyl alcohol) shaking culture behind the resuspended thalline, the beginning abduction delivering.In the fermented liquid of abduction delivering, added the methyl alcohol that final concentration is 1% (v/v) in per 24 hours.
Seven. preparing method's (technical scale) of genic mutation type recombinant protein enzyme K of the present invention
From the transformant of picking, obtain 3 the highest bacterial strains of genic mutation type recombinant protein enzyme K expression amount.Shake-flask culture three strain engineering strains, centrifugal results fermented supernatant fluid carry out enzyme and live, express tests such as output, digestion BSA, further choose the best bacterial strain of a strain and carry out the pilot scale scale-up.Carry out the engineering strain optimal culture condition 30 liters of fermentor tank levels and explore, comprise the consumption of culture medium prescription, dissolved oxygen amount, induction time and inductor methyl alcohol etc.
One preferred embodiment according to the present invention; Said pilot scale amplification test can carry out under following condition: adopt with shake flask fermentation in identical substratum; The methanol induction time is 48 ~ 168 hours, and dissolved oxygen amount is 20% ~ 40%, and the consumption of inductor methyl alcohol is 0.5% ~ 3% (v/v).
With the technical scheme that 30 liters of fermentor tanks obtain, be transplanted to 2 tons of jar industrial scale tests, duplicate the pilot scale fermentation condition fully, continuously ferment and cultivated 48 ~ 168 hours, online at any time monitoring each item fermentation index and cataphoretic determination protein expression level.
Highly preferred embodiment according to the present invention, the methanol induction time is 72 hours.
Following jar spinning thalline and fermented liquid as said genic mutation type recombinant protein enzyme K (being secreted into the proteolytic enzyme in the substratum) when being in the state that is present in the fermented liquid supernatant, can be used; Also can use said proteolytic enzyme through concentrating said fermented liquid supernatant.But the said genic mutation type recombinant protein of purifying or partial purification enzyme K (being secreted into the proteolytic enzyme in the substratum).
Utilize the general method of protein purification, can realize purifying or partial purification.For example, can use chromatogram (like IX or gel-filtration), ammonium sulfate precipitation or the organic solvent precipitation technology of comprising.Also can be through the enzyme behind the concentrated said purifying such as freeze-drying, ultra-filtration membrane and organic solvent precipitation.
One preferred embodiment according to the present invention; Be utilized in six polyhistidyl labels of the C end interpolation of genic mutation type recombinant protein enzyme K of the present invention; Fermented liquid at first passes through spinning; Supernatant is through the metal ion-chelant affinitive layer purification, and then through weak cation displacement chromatography purifying, freeze-drying becomes the finished product enzyme again; Albumen major part when ultrafiltration is precipitated out and the defective that causes yield to reduce when having avoided adopting ultrafiltration-cation seperation column-ultrafiltration-anion column to carry out purifying in traditional purification process, thereby makes comprehensive yield compared prior art improve 80%.
Embodiment
Can understand the present invention better by means of following embodiment, these embodiment only are used to illustrate the present invention, should not be interpreted as limitation of the present invention.
In addition, the carrying out that can be put down in writing by Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)) of all genetic manipulation.
The structure of embodiment 1 Proteinase K expression vector pPIC9K-ProK and pPIC9K-mutProK
The encoding sequence (SEQ ID NO.3) of wild-type protease K is replaced with the dna sequence dna that the pichia spp preference codon is formed; Add SnaB I restriction enzyme site TACGTA at its 5 ' end; 3 ' end adds six polyhistidyl label C ACCATCACCACCATCAC (SEQ ID NO.4), terminator codon TAA and NotI restriction enzyme site GCGGCCGC in order, utilizes the sequence (SEQ ID NO.5) of the synthetic gained of chemical process.The SnaB I restriction enzyme site TACGTA that 5 ' end is added replaces with EcoR I restriction enzyme site GAATTC; And further introduce corresponding sudden change back (as shown in Figure 1), utilize the sequence (SEQ ID NO.6-9) (the Canadian gene synthesis technology BioBasic Inc. of service company) of the synthetic gained of chemical process.The sequence of above-mentioned chemosynthesis all has been cloned into (the Canadian gene synthesis technology BioBasic Inc. of service company) in the pUC57 plasmid.
Amplification pUC57/ProK plasmid and pUC57/mutProK plasmid; With restriction enzyme SnaB I, Not I and EcoR I, Not I ProK and mutProK encoding sequence are downcut respectively; (American I nvitrogen company) is connected with the pPIC9K carrier that carries out same double digestion, will connect product and transform DH5 α competent cell, the picking clone; The amplification plasmid is identified plasmid with restriction enzyme SnaB I, Not I or EcoR I, Not I respectively.
Pass through aforesaid method; With the dna fragmentation subclone (as shown in Figure 2) to Yeast expression carrier pPIC9K of encoding wild type Proteinase K and genic mutation type recombinant protein enzyme K of the present invention, thereby construct wild-type protease K expression vector pPIC9K-ProK and genic mutation type recombinant protein enzyme K expression vector pPIC9K-mutProK:pPIC9K-mutProK6 of the present invention (Y151A, K208H, S273T, G293A, K332R, S337N), pPIC9K-mutProK7-1 (S123A, Y151A, K208H, S273T, G293A, K332R, S337N), pPIC9K-mutProK7-2 (Y151A, L180I, K208H, S273T, G293A, K332R, S337N) and pPIC9K-mutProK8 (S123A, Y151A, L180I, K208H, S273T, G293A, K332R, S337N) (as shown in Figure 1).
Sequencing result shows that target fragment is all correctly inserted in the above-mentioned expression vector.The conversion of embodiment 2 Proteinase K expression vector pPIC9K-ProK and pPIC9K-mutProK
Utilize restriction enzyme Sal I enzyme to cut recombinant plasmid constructed among the embodiment 1; Make it linearizing; Use TE damping fluid (pH 8.0) to be dissolved to the concentration of 1 μ g/ μ L then; Get 20 μ L dissolved plasmids and GS115 yeast competent cell mixing, be transferred in the electric revolving cup (two clearance between poles 0.1cm) of ice precooling ice bath 5min.The method that adopts electric shock to transform is utilized the built-in pichia spp of electric conversion instrument to transform parameter and is shocked by electricity, and above-mentioned expression vector is transformed in the GS115 yeast competent cell.
After electric shock finishes, in electric revolving cup, add the 1M Sorbitol Solution USP of 1mL ice precooling rapidly, mixing goes in the 1.5mL EP pipe gently.The thalline suspension is coated MD flat board (13.4g/L yeast basis nitrogenous source; 0.4mg/L vitamin H; 20g/L glucose, 1.5% agar) on, per 500 μ L coating one flat plate.Place 30 ℃ of environment to cultivate flat board,, obtain GS115/pPIC9K-ProK and GS115/pPIC9K-mutProK until single bacterium colony occurring.
Prepare the YPD screening dull and stereotyped (yeast extract 10g/L, peptone 20g/L, glucose 20g/L, 1.5% agar) of four G418 concentration gradients (0.75mg/mL, 1mg/mL, 1.75mg/mL, 2.0mg/mL) respectively.Dull and stereotyped for the MD that in embodiment 2, cultivates, with 70 yeast lists of each picking of aseptic toothpick bacterium colony, point screens in the flat board at the YPD of above-mentioned four G418 concentration gradients, and makes corresponding numbering.Each clone is all cooked the screening of 4 gradients.
The pPIC9K expression vector can be given pichia spp Geneticin resistance, and its resistance level mainly depends on the copy number of the kan gene of integration.After the pPIC9K of single copy is incorporated into the pichia spp genome, can give the Geneticin resistance level of the about 0.25mg/mL of pichia spp.Owing to have genetic linkage between kan gene and the expression cassette (pAOX1 and goal gene), thereby the target protein encoding sox copy number that can come roughly to infer this clone according to the resistance level of Geneticin and comprised.
Dull and stereotyped through observing above-mentioned screening, whether preliminary judgement GS115/pPIC9K-ProK and GS115/pPIC9K-mutProK yeast clone contain the target protein encoding sox of high copy number.The content of G418 is high more in the screening flat board, and yeast colony growth profile is big more, shows the copy that contains more target protein gene in this yeast mono-clonal, can tolerate the G418 of higher concentration.
Simultaneously, above-mentioned mono-clonal being carried out PCR identifies.Primer is 5'AOX1 (SEQ ID NO.10), 3'AOX1 (SEQ ID NO.11).
5'AOX1：GACTGGTTCCAATTGACAAGC
3'AOX1：GCAAATGGCATTCTGACATCC
PCR system (20 μ L):
The PCR program:
94℃5min；
94 ℃ of 30s, 55 ℃ of 1min, 72 ℃ of 2min 15s, 30 circulations;
72℃6min。
Utilize agarose gel electrophoresis to detect the size of PCR product.Behind Sal I linearization plasmid conversion GS115, on the HIS4 site, to recombinate mostly, most of transformants are Mut
+Phenotype; Yet because plasmid contains the AOX1 gene order, might recombinate, destroy wild-type AOX1 gene, produce Mut in the AOX1 site
STransformant.Therefore, in theory, if the purpose fragment is inserted success and phenotype is Mut
+, two bands about 2200bp, about 1632bp should be arranged; If have only band of 1632bp, then phenotype possibly be Mut
S
PCR result shows and can amplify the band about 2200bp, show that the purpose fragment all inserts success, and phenotype is Mut
+
Expression and the detection (laboratory scale) of embodiment 4 genic mutation type recombinant protein enzyme K in yeast cell
With screening high copy number and the Mut that obtains among the embodiment 3
+In the yeast list colony inoculation of the GS115/pPIC9K-ProK of phenotype and GS115/pPIC9K-mutProK to the 20mL YPD substratum (yeast extract 10g/L, peptone 20g/L, glucose 20g/L), 30 ℃, 250rpm cultivates 24h.The centrifugal collection thalline of 1500g.With 10mL MM substratum (13.4g/LYNB, 4 * 10
-4The g/L vitamin H, 5mL/L methyl alcohol) resuspended thalline.At 30 ℃, shake bottle (2L) abduction delivering under the 250rpm subsequently.From fermented liquid, took a sample, and in the fermented liquid of abduction delivering, add the methyl alcohol that final concentration is 1% (v/v) in per 4 hours in order to detection.Continuous 5 days, cultivated altogether 120 hours.
And then; Utilize 30 liters of fermentor tanks to carry out the pilot scale scale-up, with reference to the pichia spp culture scheme (www.invitrogen.com) of American I nvitrogen company, adopt with shake flask fermentation in identical substratum; With the methanol induction time set is 120 hours; Dissolved oxygen amount is controlled at 30%, and the consumption of inductor methyl alcohol is 1% (v/v), from fermented liquid, takes a sample in order to detecting in per 4 hours.
After the cultivation, centrifugal substratum, thus obtain containing the fermented liquid supernatant of said gene mutant recombinant protein enzyme K, and place-70 ℃ of preservations.
Utilize SDS-PAGE, Western blotting and Native-PAGE to detect the expression of genic mutation type recombinant protein enzyme K of the present invention in yeast cell.
1.SDS-PAGE detect
Carrying out SDS-PAGE according to following experimental program detects:
1.1 preparation SDS-PAGE gel:
1.1.1 the preparation of separation gel:
In small beaker, add required solution composition successively, pour in the gap of the double glazing unit that is pre-assembled, room temperature is placed more than the 20min, and is complete until gel polymerisation.
1.1.2 concentrate the preparation of glue:
In small beaker, add required solution composition successively, pour into the gap of the separation gel top of having condensed between double glazing unit, room temperature is placed more than the 20min, and is complete until gel polymerisation.
1.2 the supernatant of culture medium of GS115/pPIC9K-mutProK8 by-70 ℃ of taking-ups, is melted the back on ice and is handled 10min with boiling water bath.The centrifugal 1min of 12000g draws supernatant and joins in the sample duct of the gel that as above prepares, and applied sample amount is 20 μ L.Add simultaneously protein molecular weight standard (Unstained Protein Molecular Weight Marker, Fermentas).
1.3200V constant voltage electrophoresis 1.5h.
1.4 unload gel.Preparation staining fluid and destainer carry out coomassie brilliant blue staining, with the target protein band in the observation sample shown in the according to the form below.
Coomassie brilliant blue staining liquid: destainer:
The result is as shown in Figure 3, cultivates and can in fermented liquid supernatant (shake flask fermentation), observe tangible target protein band in 3.5 hours.Wherein, the sample in each swimming lane is following:
The 1st road: protein molecular weight standard;
The 2nd road: abduction delivering 51.5 hours;
The 3rd road: abduction delivering 47.5 hours;
The 4th road: abduction delivering 31.5 hours;
The 5th road: abduction delivering 27.5 hours;
The 6th road: abduction delivering 23.5 hours;
The 7th road: abduction delivering 7.5 hours;
The 8th road: abduction delivering 3.5 hours;
The 9th road: the contrast before inducing.
Wherein, (Unstained Protein Molecular Weight Marker, 7 protein bands Fermentas) represent the albumen size of 116 kD, 66.2 kD, 45 kD, 35 kD, 25 kD, 18.4 kD and 14.4 kD respectively to protein molecular weight standard.
2.Western trace
(1) transfer printing: the glue behind the electrophoresis is taken out, behind the immersion transfering buffering liquid, on shaking table, slowly shake 30min; Cut nitrocellulose filter, immerse transfering buffering liquid 5min at least; With filter paper of transferring buffered liquid wetting, be laid on the bottom electrode plate of transfer printing appearance, film is laid on the filter paper, do not stay bubble, gel is affixed on the film, do not stay bubble; Wetting another filter paper covers on gel, with the glass test tube bubble of rushing; Cover the top layer battery lead plate, connect positive and negative power supply, change film 50V, 4 ℃ and spend the night.
(2) hybridization: after changeing film, take out transfer film, room temperature, 25mL TBS washes film 5min; Transfer film immerses sealing 25mL damping fluid, incubated at room 1h; The taking-up transfer film is given a baby a bath on the third day after its birth inferior with TBST, each 5min, each 15mL; Film and an anti-diluent 10mL hybridization, 4 ℃ of concussions are spent the night; The taking-up transfer film is given a baby a bath on the third day after its birth inferior with TBST, each 5min, each 15mL; Hatch 10mL hybridization with two anti-diluents, room temperature concussion 1h; The taking-up transfer film is given a baby a bath on the third day after its birth inferior with TBST, each 5min, each 15mL; Outwell film washing liquid, film is sandwiched in middle suction of thieving paper goes excess liquid, be laid on the sheet glass; Wrap film with preservative film, do not stay bubble, on the albumen side direction, be fixed in the sheet folder and prepare exposure.
(3) develop a film: cut out egative film, compressing tablet, exposure 1-3min; Development 3min, in the water rinsing several times, photographic fixing 10 ~ 15min, flushing with clean water; Confirm the time shutter according to development effect.
The result is as shown in Figure 4, can observe tangible protein expression.Wherein, get and induce back 72 hours shake flask fermentation supernatant and 30L fermented supernatant fluid to analyze.Outside the isolating protein molecular weight standard (5 μ L), each swimming lane sample is all gone up appearance 20 μ L, and the sample in each swimming lane is following:
The 1st road: the shake flask fermentation supernatant concentrates 20 times;
The 2nd road: the shake flask fermentation supernatant concentrates 10 times;
The 3rd road: the shake flask fermentation supernatant concentrates 5 times;
The 4th road: shake flask fermentation supernatant;
The 5th road: 30L fermented supernatant fluid;
The 6th road: the 30L fermented supernatant fluid concentrates 5 times;
The 7th road: 30L fermented supernatant fluid;
The 8th road: protein molecular weight standard.
Wherein, (Precision Plus Protein Dual Color Standards, 10 protein bands Bio-Rad) represent the albumen size of 250kD, 150kD, 100kD, 75kD, 50kD, 37kD, 25kD, 20kD, 15kD, 10kD respectively to protein molecular weight standard.
The result and the GS115/mutProK8 similar (data not shown goes out) that utilize GS115/mutProK6, GS115/mutProK7-1 and GS115/mutProK7-2 to obtain.
This shows, genic mutation type recombinant protein enzyme K of the present invention can be in yeast cell effective expression.
3. native polyacrylamide gel electrophoresis (Native-PAGE)
3.1 experimental principle:
Native polyacrylamide gel electrophoresis (basic protein active electrophoresis) is a kind of ordinary method of using in the analysis of protein well-known to those skilled in the art.The low pH gel systems of this method utilization separates basic protein, accomplishes according to the molecular sieve effect of the difference of its electrophoretic mobility and gel.
3.2 instrument, reagent:
3.2.1 instrument:
Electrophoresis apparatus, whizzer, pH meter etc.
3.2.2 reagent and solution:
30% acrylamide soln: acrylic amide (Acr) 29g, N, N '-methylene bisacrylamide (Bis) 1g, adding distil water 20mL behind the mixing, 37 ℃ of dissolvings are settled to 100mL, 4 ℃ of preservations in the brown bottle.
4 * separation gel damping fluid: take by weighing 1.35g KOH, be dissolved in the 80mL water, regulate pH=4.3, add water and be settled to 100mL, in 4 ℃ of preservations with glacial acetic acid.
8 * spacer gel damping fluid: take by weighing 2.7g KOH, be dissolved in the 80mL water, regulate pH=6.8, add water and be settled to 100mL, in 4 ℃ of preservations with glacial acetic acid.
1 * electrophoretic buffer: take by weighing L-L-Ala 31.185g, add the suitable quantity of water dissolving, regulate pH=4.3 ~ 4.5, add water and be settled to 1000mL, in 4 ℃ of preservations with glacial acetic acid.
5 * sample-loading buffer: 2.5mL 8 * separation gel damping fluid, 3.625mL glycerine adds water and is settled to 10mL, and methyl green is an amount of ,-20 ℃ of preservations.
10% ammonium persulfate solution: analytical pure ammonium persulphate 1g, be dissolved in water, be settled to 10mL, in 4 ℃ of preservations.(ammonium persulphate can slowly decompose, thus every other week fresh or prepare after be placed in the little centrifuge tube ,-20 ℃ of preservations, the time spent is taken out tubule use).
TEMED (N, N, N, N-Tetramethyl Ethylene Diamine) solution: reagent stoste takes a morsel, and places brown vial, in 4 ℃ of preservations.
Coomassie brilliant blue R-250 staining fluid: Coomassie brilliant blue (Coomassie blue R-250) 0.5g, 200mL destainer.
Destainer: ethanol 450mL, Glacial acetic acid min. 99.5 100mL, water 450mL.
50mmol/L HAc-NaAc damping fluid (pH=4.0) (dilute sample is used).
3.3 working method:
3.3.1 making sheet: sheet glass is cleaned, on request sheet glass is fixed on the mould of plastics.
Prepare 15% separation gel (lower floor's glue) 10mL, fill a prescription as follows:
| Solution composition | Addition (mL) |
| Zero(ppm) water | - |
| 30% acrylamide soln | 5.05 |
| 50% glycerine | 2.27 |
| 4 * separation gel damping fluid | 2.54 |
| 8 * spacer gel damping fluid | - |
| 10% ammonium persulfate solution | 0.121 |
| TEMED solution | 0.015 |
| |
10 |
Component in the prescription is joined in the 50mL beaker, mix, use the 1mL liquid-transfering gun,, add 5.0mL in each plate along sheet glass joining in the film groove slowly.After separation gel adds, with wash bottle gently add water at Jiao Mianshang, make it water seal.Room temperature is placed about 30min, treat that glue condenses naturally after, slowly water is inclined, use the filter paper suck dry moisture.
Prepare 5% spacer gel (upper strata glue) 3mL, 5mL, fill a prescription as follows:
| Solution composition | Addition (mL) | Addition (mL) |
| Zero(ppm) water | 2.07 | 3.45 |
| 30% acrylamide soln | 0.498 | 0.83 |
| 50% glycerine | - | - |
| 4 * separation gel damping fluid | - | - |
| 8 * spacer gel damping fluid | 0.378 | 0.63 |
| 10% ammonium persulfate solution | 0.03 | 0.05 |
| TEMED solution | 0.003 | 0.005 |
| TV (mL) | 3 | 5 |
Component in the prescription is joined in the 50mL beaker, mix rapidly, slowly join along sheet glass rapidly and separate on the film, till filling it up with.Comb is inserted in the glue of upper strata gently.After treating that gelling is solid, mould is put into water, slowly transfer to comb.
3.3.2 sample preparation:
Get resulting fermented liquid 2mL, the centrifugal 1.5min of 10000r/min.Draw the sample supernatant of 80 μ L after centrifugal in little centrifuge tube with the rifle head, add 20 μ L5 * sample-loading buffers, mix.
3.3.3 last appearance:
Device is put into electrophoresis chamber, outside electrophoresis chamber, add 1 * electrophoretic buffer, in two glass clamping plate medial launders, also fill with this liquid (the appearance hole is as the criterion on the submergence adhesive tape).Carefully the sample after the 20 μ L processing is joined in the appearance hole with the rifle head.
3.3.4 electrophoresis:
With the wiring of electrophoresis apparatus, redness connects negative pole, and black connects positive pole (basic protein is used reversible circulation), connects power supply, start.Regulate output voltage to 180V.When showing that electric current is 0, the electrophoresis sample walks to till the gel bottommost.
3.3.5 dyeing:
Soak gel with coomassie brilliant blue staining liquid, under the room temperature, vibration dyeing on the shaking table.
3.3.6 decolouring:
Staining fluid is poured out,, added destainer, decolour to the sample strip clear display with the unnecessary staining fluid of zero(ppm) water flush away.
The result is referring to Fig. 5.Wherein, contrast ProK is the commercialization Proteinase K (the wild-type protease K of procaryotic cell expression) of Merck company.It is thus clear that, genic mutation type recombinant protein enzyme K of the present invention can be in yeast cell effective expression.
The active detection of embodiment 5 genic mutation type recombinant protein enzyme K
Utilize resulting shake-flask culture fermented liquid supernatant among the embodiment 4, measure protein concn, the protein concentration of fermented liquid supernatant is adjusted to 0.97mg/mL with PBS with the Bradford method.
Reaction substrate is the BSA of guanidine hydrochloride denaturation: 5mg BSA is dissolved in 6M Guanidinium hydrochloride, 50mM Tris-Cl (pH 8.0), 5mM DTT, and final volume is 1mL.95 ℃ of water-bath 20min; After reducing to room temperature, add 50mM Tris-Cl (pH 7.5) and 5mM CaCl
2, the final concentration of Guanidinium hydrochloride is reduced to below the 2M.
Temperature of reaction: 55 ℃; (100 μ L) is as shown in the table for reaction system:
| |
3 | 4 | 5 | 6 | 7 | 8 | 10 |
| BSA（μL） | 90 | 90 | 70 | 70 | 50 | 50 | 100 |
| mutProK8（μL） | 10 | 10 | 30 | 30 | 50 | 50 | 0 |
| Reaction times | 2h | Spend the night | 2h | Spend the night | 2h | Spend the night | 2h |
Reaction finishes the back and adds 25 μ L5 * sample-loading buffers, last appearance 30 μ L, 100V electrophoresis 90min.
Reset and add 25 μ L, 5 * sample-loading buffer in the sample 1:100 μ L mutProK8 fermentation, last appearance 20 μ L.
The purified spissated mutProK8 of sample 2:100 μ L adds 25 μ L, 5 * sample-loading buffer, last appearance 20 μ L.
Sample 9:10 μ L not sex change BSA adds 2.5 μ L, 5 * sample-loading buffer, last appearance 2 μ L.
Shown in Fig. 6 A, add genic mutation type recombinant protein enzyme K of the present invention after, BSA all by fully the degraded.Only containing the sample that contrasts BSA and only contain mutProK8 does not degrade.
In addition, shown in Fig. 6 B, only 0.01 μ gmutProK8, the 16 μ gBSA that just can degrade fully show that the resulting genic mutation type recombinant protein of the present invention enzyme K has outstanding activity.Wherein, sample is following in each swimming lane:
The 1st road: protein molecular weight standard;
The 2nd road: BSA 16 μ g, wild-type protease K;
The 3rd road: BSA 16 μ g, 0.01 μ g mutProK8;
The 4th road: BSA 16 μ g, 0.1 μ g mutProK8;
The 5th road: BSA 16 μ g, 1.0 μ gmutProK8.
Wherein, (Unstained Protein Molecular Weight Marker, 7 protein bands Fermentas) represent the albumen size of 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD and 14.4kD respectively to molecular weight standard.
Result and the mutProK8 that mutProK6, mutProK7-1 and mutProK7-2 obtain similar (data not shown goes out).
The above results shows that tentatively genic mutation type recombinant protein enzyme K raw product of the present invention has tangible protease activity.
The detection by quantitative of the specific activity of embodiment 6 genic mutation type recombinant protein enzyme K
Detect the enzymic activity of utilizing the genic mutation type recombinant protein enzyme K of the present invention that the experimental program among the embodiment 4 obtains through following method:
Response procedures:
With 50mM potassium phosphate solution preparation 0.65% (w/v) casein food grade solution, slowly heating is regulated pH to 7.5 with 1M HCl solution or NaOH solution until forming the single and uniform dispersion-s down at 37 ℃.Preparation contains 10mM NaAc and 5mM Ca (Ac)
2Solution, at 37 ℃ down with 0.1M HCl or NaOH adjusting pH to 7.5.Contain 10mM NaAc and 5mM Ca (Ac) with what be cooled to room temperature
2The solution compound concentration be Proteinase K (wild-type protease K (ProK) or genic mutation type recombinant protein enzyme K of the present invention (the mutProK)) solution of 0.1 ~ 0.2U/mL, this solution is for using preceding preparation.
Add each reagent (mL) successively according to following order:
Hatch 30min and manual mixing off and on for 37 ℃, with No. 50 filter paper filterings and collect clear liquor and carry out next step experiment.
Standard curve making and colorimetric reaction:
1. typical curve:
10mL forint phenol reagent is diluted with water to 40mL, preparation 0.5N F-C reagent.Slow heating is until the tyrosine dissolving and be cooled to room temperature, preparation 1.1mM L-tyrosine standard solution.
Add each reagent (mL) successively according to following order:
2. sample determination:
In 4 bottles, add each reagent (mL) successively according to following order respectively:
Hatch and manual off and on mixing, take out bottle behind the 30min and be cooled to room temperature for 37 ℃.If solution presents muddy shape, measure with helping absorbance behind the 0.45 μ m membrane filtration.Reaction solution is transferred in the Glass Containers, measures the absorbance of Std 1 ~ 4, Std blank, sample and contrast with spectrophotometer at the 660nm place.The result calculates:
1. typical curve:
△
660nmStd=A
660nmStd-A
660nmStd is blank
Use △
660nmThe amount of Std and L-tyrosine (μ mol) drawing standard graphic representation.
The gained typical curve is as shown in Figure 7.
2. sample determination:
△
660nmSample=A
660nmSample-A
660nmBlank
Utilize typical curve to calculate the amount (μ mol) of the L-tyrosine of release.
Enzymic activity (U/mL)=(amount of the L-tyrosine of release (μ mol)) * 11/ (1 * 10 * 2).
Wherein, the TV (mL) during the 11=reaction terminating
The 10=reaction times (min)
Enzyme reagent volume (mL) during the 1=reaction
Used reaction solution volume (mL) during the 2=colorimetric
U/mg solid=(U/mL enzyme)/(mg solid/mL enzyme)
U/mg albumen=(U/mL enzyme)/(mg albumen/mL enzyme)
The enzymic activity definition
1 unit of enzyme activity is defined as, and under 7.5,37 ℃ of conditions of pH, PM decomposites the L-tyrosine of 1.0 μ mol (181 μ g) from casein food grade.(forint phenol method is carried out colorimetric)
Final reagent concentration
In the reaction system of 6mL, the reagent final concentration is: 42mM potassiumphosphate, 0.54% (w/v) casein food grade, 1.7mM NaAc, 0.8mM Ca (Ac)
2Proteinase K solution with 0.1 ~ 0.2U/mL.
The result is as shown in the table:
| mutProK6 | mutProK7-1 | mutProK7-2 | mutProK8 | ProK | |
| Active (U/mg) | 38.5 | 45.0 | 43.5 | 66.5 | 31.0 |
It is thus clear that the genic mutation type recombinant protein enzyme K that the present invention prepares can demonstrate the enzymic activity that obviously is superior to neutral protease K, wherein, the mutProK8 of eight whole sudden changes of critical sites demonstrates the outstanding enzymic activity of neutral protease K enzymic activity more than 2 times; And then; Than on the basis of mutProK6, further the 123rd S being sported A (mutProK7-1) or further the 180th L being sported the enzymic activity of the recombinant protein enzyme K of I (mutProK7-2), the mutProK8 that simultaneously these two critical sites is suddenlyd change has demonstrated the enzymic activity (66.5U/mg) that significantly improves surprisingly.
Large-scale industrialization production and the purifying of embodiment 7 genic mutation type recombinant protein enzyme K
From the transformant of picking, obtain 3 the highest bacterial strains of genic mutation type recombinant protein enzyme K expression amount.Utilize the experimental program as embodiment 4 described in, shake-flask culture three strain engineering strains, purifying protein enzyme K and carry out enzyme and live, express output, digest tests such as BSA from fermented liquid further chooses the best bacterial strain of a strain and carries out the pilot scale scale-up.
Carrying out the engineering strain optimal culture condition 30 liters of fermentor tank levels explores; With reference to the pichia spp culture scheme (www.invitrogen.com) of American I nvitrogen company, adopt with shake flask fermentation in identical substratum, the methanol induction time is 120 hours; Dissolved oxygen amount is controlled at 30%; The consumption of inductor methyl alcohol is 1% (v/v), induces back sampling in per 8 hours, electrophoretic analysis Proteinase K expression amount.
With the technical scheme that 30 liters of fermentor tanks obtain, be transplanted to 2 tons of jar industrial scale tests, duplicate the pilot scale fermentation condition fully, continuously ferment and cultivated 72 hours, online at any time monitoring each item fermentation index and cataphoretic determination protein expression level.Following jar spinning thalline and fermented liquid.
The fermented liquid supernatant of getting the mutProK8 before inducing is got respectively and is induced the fermented liquid supernatant sample of back 8h, 16h, 24h, 32h, 40h, 48h, 56h, 64h to carry out the SDS-PAGE electrophoresis detection as contrast.The result is as shown in Figure 8.Wherein, sample is following in each swimming lane:
The 1st road: induce preceding contrast;
The 2nd road: induced back 8 hours;
The 3rd road: induced back 16 hours;
The 4th road: induced back 24 hours;
The 5th road: induced back 32 hours;
The 6th road: induced back 40 hours;
The 7th road: induced back 48 hours;
The 8th road: induced back 56 hours;
The 9th road: induced back 64 hours;
The 10th road: induced back 72 hours.
Result and the mutProK8 that mutProK6, mutProK7-1 and mutProK7-2 obtain similar (data not shown goes out).
It is thus clear that, utilize this large-scale industrialization mode of production, can effectively improve the productive rate (being higher than the 1.3g/L fermented liquid) of genic mutation type recombinant protein enzyme K of the present invention.This productive rate is superior to the productive rate (0.3-0.5g/L fermented liquid) of present existing Proteinase K industrial process, thereby makes later stage purifying, freeze dried high yield become possibility, for large-scale industrial production provides condition.
Be utilized in six polyhistidyl labels of the C end interpolation of recombinant protein enzyme K; Greatly reduced irrelevant foreign protein and nucleic acid and other the non-specific binding molecule of non-specific binding in the purification column; Fermented liquid is through 10; Become the finished product enzyme after the separation of 000rpm high speed centrifugation, metal-chelating column chromatography and weak cation exchange column chromatographic separation, the decolouring processing of macropore decolouring glue and the freeze-drying; Albumen major part when ultrafiltration is precipitated out and the defective that causes yield to reduce when having avoided adopting ultrafiltration-cation seperation column-ultrafiltration-anion column to carry out purifying in traditional purification process; Yield behind the purifying is higher than the initial fermented liquid of 500mg/L, thereby makes comprehensive yield compared prior art improve 80%.
Utilize SDS-PAGE and non-sex change PAGE to detect the purity of the Proteinase K after the freeze-drying of above-mentioned technology purifying.The result is as shown in Figure 9.Can find out that the recombinant protein enzyme K after the purifying freeze-drying has high purity (99%).
The stability of embodiment 8 genic mutation type recombinant protein enzyme K
The enzyme of depositing in-20 ℃ mutProK8 with the solution form and depositing in 4 ℃ mutProK8 with the lyophilized powder form stability of living is detected.
The result is shown in figure 10.Can find out that the enzyme activity of lyophilized powder remained unchanged in 1 year basically, the mutProK8 of solution form has also kept about 98.5% enzyme work after 1 year, shows and utilizes the genic mutation type recombinant protein enzyme K of method preparation of the present invention to have high stability.
What should explain at last is: above embodiment is only unrestricted in order to explanation the present invention; Although disclose preferred implementation of the present invention for illustrative purposes; Those of ordinary skill in the art is to be understood that; Can carry out various modifications, add or be equal to replacement the present invention, and not break away from the spirit and scope of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Industrial applicibility
Genic mutation type recombinant protein enzyme K using gene engineering technique mass production biologically active substance of the present invention can overcome the defective that natural product yield poorly, expense is high.The conjugated protein engineering, the molecular structure of engineered protein obtains the more powerful proteolytic enzyme product of function, and reduces cost, and these new technologies have purposes widely in industrial enzymes.
Claims (10)
1. genic mutation type recombinant protein enzyme K is characterized in that: the Y that aminoacid sequence that will be shown in SEQ ID NO.2 is the 151st sports A, the 208th K and sports H, the 273rd S and sport T, the 293rd G and sport that A, the 332nd K sport R, the 337th S sports N.
2. genic mutation type recombinant protein enzyme K as claimed in claim 1, wherein, said genic mutation type recombinant protein enzyme K also has following any one above sudden change:
(1) the 123rd S sports A;
(2) the 180th L sports I.
3. according to claim 1 or claim 2 genic mutation type recombinant protein enzyme K, wherein, the C of said genic mutation type recombinant protein enzyme K end also has six polyhistidyl labels.
4. coding is like the DNA of each described genic mutation type recombinant protein enzyme K among the claim 1-3.
5. DNA as claimed in claim 4, wherein, said DNA is all or part of of the represented nucleotide sequence of SEQ ID NO.6-9, said part has the nucleotide sequence of SEQ ID NO.6-9 7-1113 position at least.
6. comprise the expression vector like claim 4 or 5 described DNA, wherein, said expression vector is preferably pPICZ, pPICZ α, pGAPZ, pGAPZ α, pGAPZ α A and pPIC9K.
7. import the transformant of the described expression vector of claim 6; It is characterized in that; Said transformant is pichia spp (Pichia) cell, candiyeast (Candida) cell, many types of debaryomyces hansenii (Hansenula polymorpha) cell, torulopsis (Torulopsis) cell, fission yeast (Schizosaccharomyces) cell and yeast kluyveromyces fragilis (Kluyveromyces) cell; Wherein, said transformant is preferably the pichia spp cell.
8. test kit is characterized in that, said test kit comprises and is selected from the following material one or more: each described genic mutation type recombinant protein enzyme K of claim 1-3; Claim 4 or 5 described DNA; The described expression vector of claim 6; And the described transformant of claim 7.
9. preparation is like the method for each described genic mutation type recombinant protein enzyme K of claim 1-3, and said method comprises the steps: in substratum, to cultivate transformant as claimed in claim 7; And collect the said genic mutation type recombinant protein enzyme K in the said substratum.
10. method as claimed in claim 9; This method further comprises the purifying process process of said genic mutation type recombinant protein enzyme K; Said purifying process process comprises spinning, chromatographic separation, decolouring processing and freeze-drying; Wherein, said chromatographic separation is preferably and utilizes histidine-tagged metal ion-chelant affinity chromatography of carrying out.
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| CN104059900A (en) * | 2013-03-19 | 2014-09-24 | 中国科学院天津工业生物技术研究所 | Protease K and application thereof |
| CN105087529A (en) * | 2014-05-07 | 2015-11-25 | 天津旭晨科技有限公司 | Genetically engineered protease K and production method of protease K |
| CN105193640B (en) * | 2014-06-24 | 2018-10-12 | 金普诺安蛋白质工程技术（北京）有限公司 | Application of the Proteinase K in skin care and cosmetic field |
| CN105193640A (en) * | 2014-06-24 | 2015-12-30 | 金普诺安蛋白质工程技术（北京）有限公司 | Application of protease K to skin healthcare and cosmetics field |
| WO2015196829A1 (en) * | 2014-06-24 | 2015-12-30 | 金普诺安蛋白质工程技术（北京）有限公司 | Application of protease k in skin health care and cosmetic field |
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| CN109207460A (en) * | 2018-10-23 | 2019-01-15 | 大连博格林生物科技有限公司 | Recombinate muscardine Proteinase K mutant PK-M2 and preparation method |
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| CN112831487A (en) * | 2021-01-27 | 2021-05-25 | 武汉爱博泰克生物科技有限公司 | Purification method and application of recombinant proteinase K |
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| CN113215138A (en) * | 2021-06-02 | 2021-08-06 | 武汉瀚海新酶生物科技有限公司 | Proteinase K mutant with improved thermal stability |
| CN113913412A (en) * | 2021-10-13 | 2022-01-11 | 湖北大学 | Proteinase K mutant and preparation method thereof |
| CN113913412B (en) * | 2021-10-13 | 2023-10-03 | 湖北大学 | Proteinase K mutant and its preparing process |
| WO2023096513A1 (en) * | 2021-11-26 | 2023-06-01 | Blirt S.A. | Tritirachium album proteinase k mutant and its zymogen, expression plasmid, recombinant pichia pastoris strain and method of producing the mature form of proteinase k mutant |
| CN114196656A (en) * | 2021-12-29 | 2022-03-18 | 南京巨匠生物科技有限公司 | Protease K mutant and construction and application of expression vector thereof |
| CN114196656B (en) * | 2021-12-29 | 2023-10-24 | 南京巨匠生物科技有限公司 | Proteinase K mutant and construction and application of its expression vector |
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