CN104745613A - A human kallikrein, and a coding gene and an application and preparation method thereof - Google Patents
A human kallikrein, and a coding gene and an application and preparation method thereof Download PDFInfo
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Abstract
The invention provides a human tissue kallikrein-1 (hK1) coding gene, an expression and purification manner, an activity detecting method, and a modified pichia pastoris fermentation BSM culture medium. The optimized human tissue kallikrein-1 (hK1) coding gene is shown as SEQ ID NO:1. The sequence is obtained by codon optimization for a pichia pastoris expression system. Compared with natural sequences, the sequence is more conductive to hK1 expression in pichia pastoris. The established fluorescence enzyme activity detecting method is simple in operation, and can be used for screening of high-expression monoclonal recombinant engineering bacteria. In addition, the modified fermentation BSM culture medium can promote efficient expression of the hK1 protein and inhibit expression of host endogenous protein under the premise of not influencing production of the pichia pastoris, thus facilitating subsequent separation and purification. Accordingly, the coding gene, the expression and purification manner, the activity detecting method and the fermentation BSM culture medium lay foundations for expression and application of the hK1 protein in the pichia pastoris.
Description
Technical field
The invention belongs to biotechnology gene field, relate to a kind of Human kallikrein-1, encoding gene, expression and purification mode and activity test method and application thereof.
Background technology
Kallikrein (also referred to as kininogenase) is a kind of serine protease, and prokinin can be made to discharge kassinin kinin, and it has very high physiologically active, plays very important physiological action in tissue.Kallikrein is mainly divided into two classes: plasma kallikrein (Plasma Kallikrein, PK) and tissue kallikrein (Tissue Kallikrein, TK).Two kinds of enzymes are not quite similar in molecular size range, substrate specificity, amynologic characteristic and the kassinin kinin that discharges.Plasma kallikrein can participate in the surface activation of blood clot, fibrinolysis, kassinin kinin generate and inflammatory process.And tissue kallikrein is extensively present in many tissues such as kidney, pancreas, gastrointestinal tract mucosa and central nervous system, it synthesizes in the tissue with the form of prokininogenase, activate formative tissue kallikrein through trypsinase again, and can blood circulation be released into.Tissue kallikrein gene is many members family that a class is large, and family of current reference tissue kallikrein is at least made up of 15 members (KLK1-KLK15).
At present, tissue kallikrein has been applied to clinical, for the treatment of the diseases such as diabetic complication, hypertension and acute ischemic cerebral apoplexy.Comparatively common product mainly contains two classes: a class is the kallikrein (" happy open " of the such as red pharmacy in Changzhou thousand) extracted from Pancreas Sus domestica, is to originate Pancreas Sus domestica more widely for raw material extraction purification and the tissue kallikrein KLK1 that obtains.But from Pancreas Sus domestica, the process of Isolation and purification pig kininogenase is loaded down with trivial details and extracted amount is low, and due to species variation, animal source activated protein easily produces immune response in human body.Another kind of is Human Urinary Kallikrein (such as sky, Guangzhou general " Kai Likang "), it extracts primarily of in people's urine, although Human Urinary Kallikrein biological activity is apparently higher than Pancreas Sus domestica kallikrein, and eliminate the immunogenic problem of protein for animal, but still face the problem such as limited source, susceptible viral pollution.Therefore recombinant human tissue kallikrein-1(Homo sapiens kallikrein1, hK1) the efficient preparation of albumen seems very urgent, and the restructuring hK1 albumen of efficient and cheap will be obtained, must be prepared in a large number by genetic engineering technique and produce, just likely be applied in actual production.
Up to now, the engineered method of domestic and international employing utilizes yeast expression system Restruction hK1 albumen to carry out many research, but due to species variation, hK1 native sequences is difficult to directly express in yeast, and incorrect, the glycosylation modified uniformity of the normal occurrence sequence of expression product is poor, biological activity is low, yield poorly or complex manufacturing, relate to toxic reagent and be difficult to the problems such as commercial application.Such as, show two bands during the restructuring hK1 protein electrophoresis that Chan in 1998 etc. obtain, and product homogeneity (Protein Expression and Purification12,361 – 370,1998) cannot be proved; The restructuring hK1 output only secreting, expressing of about 40mg/L(human pancreas kininogenase in pichia spp that Yuan Xin in 2004 is clear etc. obtains, Yuan Xin clear Chen Jing spring, Life Science and Technology institute of Beijing University of Chemical Technology, Vol.31, No.6,2004).In addition according to Chinese patent application 200610027754.1, the Pichia anomala expression restructuring hK1 protein Process of the emerging biology research and development in Shanghai ten thousand in 2006, host Inner source protein expression amount is high, hK1 separation and purification difficulty; And adopt methyl alcohol as inductor in its technique, and methyl alcohol is dangerous organic reagent, not only cost improves, there is potential safety hazard, and also may face the problems such as methyl alcohol remains in albumen later-period purification.In addition, the emerging biology in Shanghai ten thousand with people's kidney cDNA library of Panomics company for template, obtain the gene of people hK1 by PCR method, its 162 site may exist Glu/Lys two seed amino acid, this amino acid whose polymorphism and the sugar-modified diversity of yeast expression result in the heterogeneity of product.Secondly, the activity methods of current detection hK1 mainly utilizes hK1 specific for hydrolysis chromophoric substrate S-2266(Val-Leu-Arg-pNA) chemical bond in molecule between-Arg-pNA and discharge pNA, free pNA is utilized to carry out more active size in A405 value, but the background that the expression of substratum and pichia spp intrinsic protein causes is very high, thus make this survey scheme accuracy alive very low.
In addition, that the emerging biology in Shanghai ten thousand adopts when cultivating Pichia anomala expression restructuring hK1 is industrial conventional fermention medium BSM, containing very high salt concn in this substratum, as more than pH>5, a lot of precipitation can be produced in BSM substratum, be unfavorable for thalli growth; And salt concn in substratum is too high, causes high osmotic pressure to cause in fermenting process and cause aqtocytolysis to produce lipid material and increase the residual probability of foreign DNA, for the work such as subsequent downstream purifying bring a lot of trouble, and easily make the albumen of expression precipitate.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art part, the invention provides that a kind of production technique is simpler, output is higher, and the expressing gene of expressing the more homogeneous expression system of product and adapting with it, and the measuring method for activity of hK1 and purification process.
An object of the present invention is to provide a kind of restructuring hK1 albumen, its aminoacid sequence is as shown in SEQ ID NO:2.
Another object of the present invention is to provide a kind of gene of restructuring hK1 albumen described above of encoding, and its base sequence is as shown in SEQID NO:1.This sequence carries out the codon optimized sequence obtained for pichia yeast expression system, and compared with native sequences, it is more conducive to hK1 and expresses in pichia spp.
Preferably, the present invention also added secreting signal peptide before coding restructuring hK1 gene described above, and preferably, secretion signal peptide sequence of the present invention aims at pichia spp secreting, expressing to extracellular signal peptide; More preferably, secretion signal peptide sequence of the present invention is the signal peptide sequence obtained for pichia yeast expression system optimization, significantly improves the secernment efficiency of heterologous gene in Host Strains by contrast.The base sequence of the secreting signal peptide after optimization is as shown in SEQ ID NO:3.
Another object of the present invention is to provide the carrier of a kind of gene containing above-mentioned coding restructuring hK1 or the gene with the coding restructuring hK1 of secreting signal peptide, preferably, described carrier is pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZ α A, B, C or pGAPZ α A, B, C, be more preferably pPIC3.5K, pPICZ α A or pGAPZ α A.
Another object of the present invention is to provide a kind of Pichi strain comprising carrier described above, and preferably, described Pichi strain is SMD1168, GS115, KM71, X-33 or KM71H, is more preferably KM71 or X-33 bacterial strain.
Another object of the present invention is to provide a kind of expression method of hK1 albumen of recombinating, and described method comprises following step:
A. the carrier of the gene containing coding restructuring hK1 described above or the gene with the coding restructuring hK1 of secreting signal peptide is built;
B. proceed to after the vector linearization of steps A in Pichi strain, and cultivate under suitable conditions;
C. protein purification is reclaimed.
Carrier described above is preferably pPIC3.5K, pPICZ α A and pGAPZ α A.
Pichi strain described above is preferably KM71 or X-33 bacterial strain.
Preferred, carrier described above is pGAPZ α, and Pichi strain described above is X-33 bacterial strain.
Another object of the present invention is to provide a kind of restructuring hK1 proteolytic enzyme kinetic activity detection method, and described method comprises following step:
A. first fluorogenic substrate Z-Phe-Arg-MCA is dissolved in DMSO, then is diluted to reaction density with reaction buffer;
B. the substratum containing hK1 albumen is added in enzyme plate, 2 multiple holes, then add fluorogenic substrate Z-Phe-Arg-MCA respectively.After vibration mixing, arranging excitation wavelength is 360-400nm, emission wavelength 440-480nm, and plate is read in circulation;
C. with fluorescent signal value for ordinate zou, reading the plate time is X-coordinate, and slope is fluorescent signal value and reads the ratio of plate time, and calculate the slope in each hole, slope larger hK1 protein vigor is higher.
HK1 enzyme kinetics activity test method provided by the invention may be used for mono-clonal pichia pastoris engineered strain or the cell strain of high flux screening high expression level hK1, and sensitivity and accuracy are all better than chemiluminescence method.
Another object of the present invention is to provide a kind of fermentation improvement BSM substratum of hK1 albumen high expression of recombinating, described improvement fermentation BSM culture medium prescription and being prepared as follows:
Improvement fermentation BSM substratum: glycerine 40g/L, H
3pO
49mL/L, CaSO
42H
2o0.3g/L, K
2sO
46.07g/L, MgSO
47H
2o4.97g/L, KOH1.38g/L.121 DEG C of sterilizing 20min, after temperature drops to 50 DEG C, adjust pH=6 with strong aqua.
Trace salts solution PMT1:CuSO
46.0g/L, KI0.8g/L, MnSO
4h
2o3.0g/L, Na
2moO
42H
2o0.2g/L, H
3bO
30.2g/L, CaSO
42H
2o0.5g/L, ZnCl
220g/L, FeSO
47H
2o65g/L, Biotin0.2g/L, vitriol oil 5mL/L.Filtration sterilization, 2mL/L adds in improvement fermentation BSM substratum.
As preferably, described fermentation BSM substratum strong aqua regulates pH to be 6.0-7.0.
Another object of the present invention is to provide a kind of restructuring hK1 method for purifying proteins, and described purification process is as follows:
A. will to recombinate hK1 fermented liquid low-temperature and high-speed collected by centrifugation supernatant, to add high density (NH)
2sO
4solution, makes its final concentration in supernatant be 1.0mol/L, membrane filtration.
B. first use equilibration buffer pillar, the restructuring hK1 fermented liquid then using purification system pre-treatment in steps A to be obtained passes through prepacked column or separating filler, then uses elution buffer progressively to successively decrease (NH) according to 0.2mol/L
2sO
4concentration isocratic elution, collects each elution peak, namely obtains the restructuring hK1 albumen that glycosylation modified degree is different.Described level pad contains 20mmol/L sodium phosphate, 1.0mol/L (NH)
2sO
4, 10% glycerine (v/v), pH6.0; Described elution buffer contains 20mmol/L phosphoric acid salt, 10% glycerine (v/v), pH6.0.
The purifying process of above-mentioned purifying process hK1 is mainly hydrophobic chromatography method, and prepacked column and filler are preferably phenyl.Purifying process provided by the present invention, prepares in hK1 fermented liquid in pichia yeast expression system, is more suitable for being separated the different hK1 albumen of glycosylation modified degree.
Technique effect of the present invention mainly contains:
First, the gene order through optimised restructuring hK1 albumen of the present invention, is more suitable for the expression at pichia yeast expression system, the expression amount of expressed restructuring hK1 albumen far above hK1 native gene sequence at different pichia yeast expression system.Recombinate in the pichia spp shake-flask culture scale expression amount of hK1 albumen of hK1 gene after optimization just can reach 200mg/L, according to rationally calculating that can determine to amplify after fermentation can more than 1g/L, far above expression amount of the prior art.And, contriver finds to adopt pichia spp non-methanol using type X-33 engineering bacterium expression restructuring hK1 protein Process not only not need to utilize conventional inductor methyl alcohol unexpectedly, eliminate potential safety hazard, be beneficial to the separation and purification of downstream target protein, and from prior art and other phenotype yeast expressions, to there are two kinds of glycosylation modified kallikreins different, its target protein is sugar-modified single, and product band is single, and product homogeneity is higher.
Secondly, the present invention is discharged MCA according to the chemical bond energy in fluorogenic substrate Z-Phe-Arg-MCA molecule between-Arg-MCA by hK1 specific for hydrolysis, and free MCA, at excitation wavelength 360-400nm, under emission wavelength 440-480nm, produces fluorescent signal value.Thus utilize the MCA of release in unit time amount (fluorescent signal value) be directly proportional to the activity of enzyme and measure the activity of hK1.This method avoid that background that the expression due to substratum and pichia spp intrinsic protein causes is high and accuracy that is that cause is on the low side, and the sensitivity of fluorescent signal is far away higher than absorption value, hK1 can be applied at pichia spp, high expression level monoclonal cell strain screening in the expression systems such as CHO.
Improvement fermentation BSM substratum of the present invention is optimized and obtains on fermention medium BSM basis.The present invention is through the BSM substratum of improvement, be more suitable for not isophenic restructuring hK1 engineering strain in the present invention to express, in this substratum, find do not affect the growth of cell and add the expression of hK1 albumen, expressed restructuring hK1 protein content is not only far above the basal fermentation medium BSM of unmodified, and secretion host intrinsic protein is less, downstream purification of being more convenient for.
In sum, the purification process of the fermentation BSM substratum of the restructuring hK1 gene order after optimization provided by the invention, restructuring hK1 pichia yeast expression system, improvement, enzyme activity determination method, high expression level mono-clonal screening method and restructuring hK1 is more suitable for the application of Pichia anomala expression restructuring hK1 albumen in actual industrial production.
Accompanying drawing explanation
Fig. 1 represents codon optimized front and back restructuring hK1 nucleotide sequence comparison.
The behavior hK1 natural gene nucleotide sequence that wherein original series is corresponding, i.e. codon optimized front sequence; The gene nucleotide series of behavior corresponding to majorizing sequence restructuring of the present invention hK1, the sequence after namely codon optimized.
Fig. 2-a, 2-b are codon optimized front and back restructuring hK1 gene CAI index in pichia yeast expression system.
Wherein, Fig. 2-a represents that hK1 natural gene nucleotides sequence is listed in CAI index in pichia yeast expression system is 0.64 through program computation; Restructuring hK1 codon of the present invention CAI index in pichia yeast expression system after Fig. 2-b represents optimization is 0.83 through program computation.
Fig. 3-a, 3-b are codon optimized front and back restructuring hK1 gene optimal codon frequency distribution areal map in Pichia anomala expression host.
Wherein Fig. 3-a represents that hK1 natural gene nucleotides sequence is listed in optimal codon frequency distribution areal map in Bichi yeast system, as can be seen from the figure: the poor efficiency codon of hK1 natural gene nucleotide sequence occurs that per-cent is 7%; Fig. 3-b represents the restructuring hK1 codon of the present invention optimal codon frequency distribution areal map in Bichi yeast system after optimization, and the restructuring hK1 Codon sequences poor efficiency codon of the present invention after optimization appears as 0.
Fig. 4-a, 4-b are codon optimized front and back restructuring hK1 gene average GC base contents distributed areas figure in pichia yeast expression system.
Wherein, Fig. 4-a represents that hK1 natural gene nucleotides sequence is listed in average GC base contents in pichia yeast expression system and is: 56.04%; Restructuring hK1 codon of the present invention average GC base contents in pichia yeast expression system after Fig. 4-b represents optimization is: 44.56%.
Fig. 5-a, 5-b are the secondary structure prediction figure of codon optimized front and back restructuring hK1mRNA.
The secondary structure prediction figure of Fig. 5-a hK1 natural gene mRNA, Fig. 5-b be codon optimized after of the present invention restructuring hK1mRNA secondary structure prediction figure.
Fig. 6-a, 6-b, 6-c, 6-d are codon optimized front and back restructuring hK1 different expression plasmid building process figure.
Fig. 6-a is restructuring hK1 natural gene expression plasmid pPIC3.5K-hK1 building process figure; Fig. 6-b is the codon optimized rear expression plasmid pPIC3.5K-opt-hK1 building process figure of restructuring hK1; Fig. 6-c is the codon optimized rear expression plasmid pPICZ α-opt-hK1 building process figure of restructuring hK1; Fig. 6-d is the codon optimized rear expression plasmid pGAPZ α-opt-hK1 building process figure of restructuring hK1.
Fig. 7-a, 7-b are the expression identification figure that the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes in slow type KM71 host engineering bacteria.In figure, arrow indication is restructuring hK1 albumen.
Fig. 7-a is after the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes slow type KM71 host engineering strain to express one week by methanol induction, bacterium liquid supernatant SDS-PAGE gel electrophoresis figure.Wherein swimming lane 1 is the pre-dyed albumen loading Marker(SM1811 of 10-250KD scope, purchased from Fermentas); Swimming lane 2-7 is that each positive monoclonal host engineering strain containing codon optimized rear restructuring hK1 gene screened by G418 cultivates bacterium liquid supernatant.
Fig. 7-b is after the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes slow type KM71 host engineering strain to express one week by methanol induction, bacterium liquid supernatant protein western blot figure.Wherein swimming lane 1 is the pre-dyed albumen loading Marker of 10-250KD scope; Swimming lane 2-7 is that each positive monoclonal host engineering strain containing codon optimized rear restructuring hK1 gene screened by G418 cultivates bacterium liquid supernatant.
Fig. 8-a, 8-b are the expression identification figure that the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes in Quick-type X-33 host engineering bacteria.In figure, arrow indication is restructuring hK1 albumen.
Fig. 8-a is after the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes Quick-type X-33 host engineering strain to express one week by methanol induction, bacterium liquid supernatant SDS-PAGE gel electrophoresis figure.Wherein swimming lane 1 is the pre-dyed albumen loading Marker of 10-250KD scope; Swimming lane 2-7 is that each positive monoclonal host engineering strain containing codon optimized rear restructuring hK1 gene screened by Zeocin cultivates bacterium liquid supernatant.
Fig. 8-b is after the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes Quick-type X-33 host engineering strain to express one week by methanol induction, bacterium liquid supernatant protein western blot figure.Wherein swimming lane 1 is the pre-dyed albumen loading Marker of 10-250KD scope; Swimming lane 2-7 is that each positive monoclonal host engineering strain containing codon optimized rear restructuring hK1 gene screened by Zeocin cultivates bacterium liquid supernatant.
Fig. 9-a, 9-b are the expression identification figure in the non-methanol using type X-33 host engineering bacteria containing codon optimized rear restructuring hK1 gene.In figure, arrow indication is restructuring hK1 albumen.
Fig. 9-a is after the non-methanol using type X-33 host engineering strain containing codon optimized rear restructuring hK1 gene expresses one week, bacterium liquid supernatant SDS-PAGE gel electrophoresis figure.Wherein, swimming lane 1 is the pre-dyed albumen loading Marker of 10-250KD scope.Swimming lane 2-7 is that each positive monoclonal host engineering strain containing codon optimized rear restructuring hK1 gene screened by Zeocin cultivates bacterium liquid supernatant, and the restructuring hK1 albumen that this system is expressed as seen is single band, illustrates that product uniformity is higher;
Fig. 9-b is after the non-methanol using type X-33 host engineering strain containing codon optimized rear restructuring hK1 gene expresses one week, bacterium liquid supernatant protein western blot figure.Wherein swimming lane 1 is the pre-dyed albumen loading Marker of 10-250KD scope; Swimming lane 2-7 is that each positive monoclonal host engineering strain containing codon optimized rear restructuring hK1 gene screened by Zeocin cultivates bacterium liquid supernatant.
Figure 10-a, the 10-b methyl alcohol be respectively containing natural hK1 gene and codon optimized rear restructuring hK1 gene to utilize in slow type KM71 host engineering bacteria abduction delivering after one week, bacterium liquid supernatant SDS-PAGE gel electrophoresis qualification figure.In figure, arrow indication is restructuring hK1 albumen.
Wherein, Figure 10-a is after the methyl alcohol containing natural hK1 gene utilizes slow type KM71 host engineering strain to express one week, bacterium liquid supernatant SDS-PAGE gel electrophoresis figure.Swimming lane 1 is the pre-dyed albumen loading Marker of 10-250KD scope, swimming lane 2-12 is that the natural hK1 gene each positive monoclonal host engineering strain that contains screened by G418 cultivates bacterium liquid supernatant.
Figure 10-b is after the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes slow type KM71 host engineering strain to express one week, bacterium liquid supernatant SDS-PAGE gel electrophoresis figure.Swimming lane 3 is the pre-dyed albumen loading M of 10-250KD scope
ark
ethe each positive monoclonal host engineering strain containing codon optimized rear restructuring hK1 gene that r, swimming lane 1-2 and 4-12 are screened by G418 cultivates bacterium liquid supernatant.Visible, utilize the slow type KM71 host engineering strain expression amount methyl alcohol be significantly higher than containing natural hK1 gene to utilize slow type KM71 host engineering strain containing codon optimized rear restructuring hK1 gene de methyl alcohol.
Figure 11-a, the 11-b methyl alcohol be respectively containing natural hK1 gene and codon optimized rear restructuring hK1 gene utilizes in slow type KM71 host engineering bacteria that abduction delivering is after one week, and hK1 protein-active of recombinating in bacterium liquid supernatant measures and compares histogram.
Wherein, Figure 11-a is that the methyl alcohol containing natural hK1 gene utilizes each mono-clonal engineering strain of slow type KM71 host, through methanol induction after one week, and the bacterium liquid supernatant relative reactivity histogram of the restructuring hK1 albumen of variable expression.X-coordinate is the restructuring hK1 albumen that the methyl alcohol containing original hK1 gene utilizes the different mono-clonal bacterial strain expression of slow type KM71 host; Ordinate zou is relative reactivity.
Figure 11-b is that the methyl alcohol containing the restructuring hK1 gene after codon optimized utilizes each mono-clonal engineering strain of slow type KM71 host, through methanol induction after one week, and the bacterium liquid supernatant relative reactivity histogram of the restructuring hK1 albumen of variable expression.X-coordinate is that the methyl alcohol containing the restructuring hK1 gene after codon optimized slowly utilizes the different mono-clonal engineering strain of type KM71 host to express restructuring hK1 albumen; Ordinate zou is relative reactivity.By more known, under same culture conditions, the albumen Bioactivity that methyl alcohol containing the restructuring hK1 gene after codon optimized utilizes slow type KM71 host engineering strain to utilize slow type KM71 host engineering strain to express far above the methyl alcohol containing natural hK1 gene, illustrates that the methyl alcohol containing the restructuring hK1 gene after codon optimized utilizes slow type KM71 host engineering strain expression amount to utilize slow type KM71 host engineering strain far above the methyl alcohol containing natural hK1 gene.
Figure 12 ferments in BSM substratum containing host's engineering strain that codon optimized front and back restructuring hK1 gene is different at basal fermentation BSM substratum and improvement, different time bacterium liquid supernatant SDS-PAGE gel electrophoresis figure.In figure, arrow indication is restructuring hK1 albumen.
Wherein, Figure 12-a is that the methyl alcohol containing natural hK1 gene utilizes slow type KM71 host engineering strain in basal fermentation BSM substratum and improvement fermentation BSM substratum, different induction time bacterium liquid supernatant SDS-PAGE gel electrophoresis figure, swimming lane 1 be the pre-dyed albumen loading Marker of 10-250KD scope, swimming lane 2-7 be respectively for the methyl alcohol containing natural hK1 gene utilize slow type KM71 host engineering strain to induce 48h, 72h, 96h, 120h, 144h, 168h in basal fermentation BSM substratum after bacterium liquid supernatant; Swimming lane 8-13 be respectively for the methyl alcohol containing natural hK1 gene utilize slow type KM71 host engineering strain to induce 48h, 72h, 96h, 120h, 144h, 168h in improvement fermentation BSM substratum after bacterium liquid supernatant.
Figure 12-b utilizes slow type KM71 host engineering strain at basal fermentation BSM substratum for the methyl alcohol containing codon optimized rear restructuring hK1 gene and improves in fermentation BSM substratum, different induction time bacterium liquid supernatant SDS-PAGE gel electrophoresis figure, swimming lane 1 is bacterium liquid supernatant after the pre-dyed albumen loading Marker of 10-250KD scope, the swimming lane 2-7 methyl alcohol be respectively containing codon optimized rear restructuring hK1 gene utilizes slow type KM71 host engineering strain to induce 48h, 72h, 96h, 120h, 144h, 168h in basal fermentation BSM substratum; Bacterium liquid supernatant after the swimming lane 8-13 methyl alcohol be respectively containing codon optimized rear restructuring hK1 gene utilizes slow type KM71 host engineering strain to induce 48h, 72h, 96h, 120h, 144h, 168h in improvement fermentation BSM substratum.
Figure 12-c is that the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes Quick-type X-33 host engineering strain in basal fermentation BSM substratum and improvement fermentation BSM substratum, different induction time bacterium liquid supernatant SDS-PAGE gel electrophoresis figure, swimming lane 1 be the pre-dyed albumen loading Marker of 10-250KD scope, swimming lane 2-7 respectively containing codon optimized rear restructuring hK1 gene methyl alcohol utilize Quick-type X-33 host engineering strain to induce 48h, 72h, 96h, 120h, 144h, 168h in basal fermentation BSM substratum after bacterium liquid supernatant; Bacterium liquid supernatant after the swimming lane 8-13 methyl alcohol be respectively containing codon optimized rear restructuring hK1 gene utilizes Quick-type X-33 host engineering strain to induce 48h, 72h, 96h, 120h, 144h, 168h in improvement fermentation BSM substratum.
Figure 12-d is that the non-methanol using type X-33 host engineering strain containing codon optimized rear restructuring hK1 gene ferments in BSM substratum at basal fermentation BSM substratum and improvement, different induction time bacterium liquid supernatant SDS-PAGE gel electrophoresis figure, swimming lane 1 is bacterium liquid supernatant after the pre-dyed albumen loading Marker of 10-250KD scope, the swimming lane 2-7 non-methanol using type X-33 host engineering strain be respectively containing codon optimized rear restructuring hK1 gene induces 48h, 72h, 96h, 120h, 144h, 168h in basal fermentation BSM substratum; Bacterium liquid supernatant after the swimming lane 8-13 non-methanol using type X-33 host engineering strain be respectively containing codon optimized rear restructuring hK1 gene induces 48h, 72h, 96h, 120h, 144h, 168h in improvement fermentation BSM substratum.According to Figure 12, no matter whether codon optimized, host's engineering strain expression amount in improvement fermentation BSM substratum of the restructuring hK1 containing different expression vector is all obviously better than traditional infrastructure fermentation BSM substratum, and assorted band is less.
Figure 13 is that the host's engineering strain containing codon optimized rear restructuring hK1 gene ferments in BSM substratum in the improvement of different pH value, is expressed, bacterium liquid supernatant SDS-PAGE gel electrophoresis figure by methanol induction.In figure, arrow indication is restructuring hK1 albumen.
Wherein swimming lane 1 be the pre-dyed albumen loading Marker of 10-250KD scope, swimming lane 2-6 be respectively host's engineering strain containing codon optimized rear restructuring hK1 gene pH=4.0,5.0,6.0,7.0, the improvement fermentation BSM substratum methanol induction of 8.0 expresses bacterium liquid supernatant.As seen from the figure, be comparatively beneficial to the expression of restructuring hK1 albumen under the condition of neutral slant acidity, the optimal pH that the host's engineering strain containing codon optimized rear restructuring hK1 gene is expressed in improvement fermentation BSM substratum is 6.0-7.0.
Figure 14 is that the host's engineering strain containing codon optimized rear restructuring hK1 gene ferments in BSM substratum in improvement, the bacterium liquid supernatant SDS-PAGE gel electrophoresis figure of different cell density and methanol induction concentration.In figure, arrow indication is restructuring hK1 albumen.
Wherein, swimming lane 1 is the pre-dyed albumen loading Marker of 10-250KD scope; Swimming lane 2 is cell densities is 15g/L, and methanol induction concentration is the bacterium liquid supernatant of 0.5%; Swimming lane 3 is cell densities is 30g/L, and methanol induction concentration is the bacterium liquid supernatant of 0.5%; Swimming lane 4 is cell densities is 60g/L, and methanol induction concentration is the bacterium liquid supernatant of 0.5%; Swimming lane 5 is cell densities is 120g/L, and methanol induction concentration is the bacterium liquid supernatant of 0.5%; Swimming lane 6 is cell densities is 15g/L, and methanol induction concentration is the bacterium liquid supernatant of 1.0%; Swimming lane 7 is cell densities is 30g/L, and methanol induction concentration is the bacterium liquid supernatant of 1.0%; Swimming lane 8 is cell densities is 60g/L, and methanol induction concentration is the bacterium liquid supernatant of 1.0%; Swimming lane 9 is cell densities is 120g/L, and methanol induction concentration is the bacterium liquid supernatant of 1.0%; Swimming lane 10 is cell densities is 15g/L, and methanol induction concentration is the bacterium liquid supernatant of 1.5%; Swimming lane 11 is cell densities is 30g/L, and methanol induction concentration is the bacterium liquid supernatant of 1.5%; Swimming lane 12 is cell densities is 60g/L, and methanol induction concentration is the bacterium liquid supernatant of 1.5%; Swimming lane 13 is cell densities is 120g/L, and methanol induction concentration is the bacterium liquid supernatant of 1.5%.
Figure 15 is restructuring hK1 fermentation liquor AKTA
tMthe hydrophobic purifying color atlas of avant150.
Wherein, Peak1 is the restructuring hK1 albumen that molecular weight is higher, and Peak2 is molecular weight higher and lower restructuring hK1 mixture, and Peak3 is the restructuring hK1 albumen that molecular weight is lower.
Figure 16 is restructuring hK1 albumen HisTrap phenyl HP(17-5195-01, purchased from GE healthcare), SDS-PAGE electrophorogram after prepacked column purifying.In figure, arrow indication is restructuring hK1 albumen.
Wherein, swimming lane 1 is the pre-dyed albumen loading Marker of 10-250KD scope; Swimming lane 2 is collection tube samples before Peak1 peak, swimming lane 3 is Peak1 peak point collection tube samples, swimming lane 4 is Peak1 tail of the peak collection tube samples, swimming lane 5 is collection tube samples before Peak2 peak, swimming lane 6 is Peak2 peak point collection tube samples, and swimming lane 7 is Peak2 tail of the peak collection tube samples, and swimming lane 8 is collection tube samples before Peak3 peak, swimming lane 9 is Peak3 peak point collection tube samples, and swimming lane 10 is Peak3 tail of the peak collection tube samples.
Figure 17-a, 17-b are that in benzene second, acyl sulphur amino acid (PTH-AA) hybrid standard product and restructuring hK1 albumen n end analyze chromatogram collection of illustrative plates
Wherein, Figure 17-a is acyl sulphur amino acid (PTH-AA) hybrid standard product (163-12271, Wako) color atlas in benzene second, and Figure 17-b is restructuring hK1 albumen n end residue analysis color atlas.After 5 spectrograms represent and carry out 5 loop tests, the order-checking spectrogram corresponding to N terminal amino acid, is followed successively by Ile, Val, Gly, Gly, Trp, completely the same with native sequences.
Figure 18-a, 18-b, 18-c are MALDI standard substance and restructuring hK1 albumen relative molecular mass test mass spectrum
Wherein, Figure 18-a is MALDI standard substance (ProteoMassTM Peptide & Protein MALDI-MSCalibration Kit, MSCAL1, Sigma-Aldrich) mass spectrum, Figure 18-b is the restructuring hK1 Protein map that relative molecular weight is higher, and Figure 18-c is the restructuring hK1 Protein map that relative molecular weight is lower.From measurement result, the higher restructuring hK1 albumen relative molecular mass size of relative molecular mass is 30928Da, and the restructuring hK1 albumen relative molecular mass size that relative molecular mass is on the low side is 28511.8Da.
Figure 19 is the point focusing electrophorogram such as isoelectrofocusing (IEF) standard substance and restructuring hK1.In figure, arrow indication is restructuring hK1 albumen.
Wherein, swimming lane 1,3 is that pI is from 4.45-9.6 isoelectrofocusing standard substance (161-0310, Bio-Rad); Swimming lane 2 is the restructuring hK1 albumen that relative molecular mass is higher; Swimming lane 4 is the restructuring hK1 albumen that relative molecular mass is lower.Result shows, and the restructuring hK1 albumen pI that the relative molecular mass of Pichia anomala expression is higher and on the low side is all about 4.40.
Figure 20 is the extreme ultraviolet CD scanning spectra of restructuring hK1 albumen, and result shows, and the secondary structure of the restructuring hK1 that the relative molecular mass of Pichia anomala expression is higher and on the low side does not have difference substantially.
Figure 21 is that SDS-PAGE gel electrophoresis figure is analyzed in the de-glycosylation of restructuring hK1 albumen.In figure, arrow indication is restructuring hK1 albumen.Result shows, and the molecular size range after the restructuring hK1 albumen removal that relative molecular mass quality is higher and on the low side is glycosylation modified is basically identical.
Wherein, swimming lane 1 is the restructuring hK1 albumen that relative molecular mass is larger, and swimming lane 2 is the restructuring hK1 albumen that relative molecular mass is less; Swimming lane 3 is the pre-dyed albumen loading Marker of 10-250KD scope; Swimming lane 4 is N-glycosidase F(11365185001, purchased from Roche) restructuring hK1 albumen de-glycosylation that Glycosylase is larger to relative molecular mass, swimming lane 5 is the restructuring hK1 albumen de-glycosylation that N-glycosidase F Glycosylase is less to relative molecular mass; Swimming lane 6 is the restructuring hK1 albumen at expression in escherichia coli.
Figure 22-a, 22-b are Recombinant Swine kallikrein and restructuring hK1 proteolytic enzyme kinetic activity mensuration figure
Wherein, the restructuring hK1 enzyme kinetics determination of activity figure of Figure 22-a to be positive control pig kallikrein enzyme kinetics determination of activity figure, Figure 22-b be different relative molecular mass.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further, should be understood that quoting embodiment is only not used in for illustration of the present invention and limits the scope of the invention.
embodiment 1 recombinate hK1 gene optimization design
Contriver is according to GenBank published Human kallikrein 1(Homo sapiens kallikrein1) cDNA sequence (GenBank accession number: BC005313) (without codon optimized gene, natural gene) and published Human kallikrein 1(Homo sapiens kallikrein) aminoacid sequence (GenBank accession number: AAA59455.1), to this gene carry out codon optimized after obtain restructuring hK1 gene of the present invention, as shown in SEQ ID No:1.Here is carried out codon optimized to restructuring hK1, and before and after optimizing, each parameter comparison is described as follows:
1. codon adaptation indexI (CAI)
From Fig. 2-a, before codon is not optimized, by calculating, restructuring hK1 natural gene codon adaptation indexI (CAI) in pichia yeast expression system is 0.64.From Fig. 2-b, by codon optimized, restructuring hK1 gene CAI index in pichia yeast expression system is 0.83.Be considered to this gene during usual CAI=1 is optimal high expression state in this expression system, CAI index is lower shows that this gene expression level in this host is poorer, therefore can find out have passed through codon optimized after the gene order that obtains can improve the expression level of restructuring hK1 gene in pichia yeast expression system.
2. optimal codon frequency of utilization (FOP)
From Fig. 3-a, based on yeast expression vector, before codon is not optimized, the poor efficiency codon of the hK1 native gene sequence codon of 30% (utilization ratio lower than) occurs that per-cent is 7%.The natural gene that this is not optimized adopts series connection rare codon, and these codons may reduce translation efficiency, even can dismiss translation assemblage.From Fig. 3-b, after codon optimized, restructuring hK1 gene occurs that in Bichi yeast system the frequency of poor efficiency codon is 0.
3.GC base contents (GC curve)
GC content ideal distribution region is 30%-70%, all can affect to some extent transcribe and translation efficiency at this any peak of extra-regional appearance.Contrasted from the GC base average content distributed areas figure of the hK1 gene of Fig. 4-a, Fig. 4-b, be 56.04% by showing hK1 natural gene GC base average content in Fig. 4-a, optimize the rear GC content peak value removed 60bp and occur outside 30%-70% region by demonstrating in Fig. 4-b, the GC base average content of the hK1 that recombinates after being finally optimized is 44.56%.
4. before and after optimizing, cis-acting elements situation is as follows:
| Cis-acting elements | Before optimization | After optimization |
| Splice(GGTAAG) | 0 | 0 |
| Splice(GGTGAT) | 1 | 0 |
| PolyA(AATAAA) | 0 | 0 |
| PolyA(AAAAAA) | 0 | 0 |
| Destabilizing(ATTTA) | 1 | 0 |
| PolyT(TTTTTT) | 0 | 0 |
5. optimize before and after the palindrome and tumor-necrosis factor glycoproteins situation as follows:
The secondary structure prediction figure of 6.mRNA
After DNA is transcribed into mRNA, because mRNA is strand linear molecule, by folded back on itself, complementary base pair is met, by the hairpin structure (Hairpin) of hydrogen bonded.5 ' hairpin structure can play regulating and controlling effect in the translation initiation stage.If but hairpin structure is very long, the required energy that unwinds is very high, just likely has influence on translation.So need the sequence expressed should avoid long and that energy is high hairpin structure as far as possible.After codon optimized, from the secondary structure prediction figure of the codon optimized front and back mRNA of Fig. 5-a, Fig. 5-b hK1,5 ' hairpin structure after optimization is reasonable with the required energy that unwinds.
embodiment 2: the expression plasmid of restructuring hK1 gene builds
1. the fragment of the restructuring hK1 full genome synthesis of front and back will be optimized respectively, be building up in pUC57 plasmid (purchased from Nanjing Jin Sirui Science and Technology Ltd.), obtain one and preserve plasmid for a long time, the plasmid before optimizing is designated as pUC57-hK1, and the plasmid after optimization is designated as pUC57-opt-hK1 plasmid.
2. pUC57-hK1 and the pUC57-opt-hK1 plasmid obtained in step 1 is used EcoR I(R0101S respectively, purchased from New England Biolabs company) and Not I(R0189S, purchased from New England Biolabs company) after double digestion, 1% agarose electrophoresis, the gene product DNA gel obtained reclaims test kit (DP214, Beijing Tian Gen biochemical technology company limited) purifying.With T4 ligase enzyme (M0202S, purchased from New England Biolabs) be connected to pPIC3.5K plasmid (V173-20, purchased from Invitrogen company) in, be transformed into DH5 α competent cell (CB101, purchased from Beijing Tian Gen biochemical technology company limited) in, 37 DEG C of overnight incubation in the LB solid medium containing penbritin (purchased from Amresco company).Picking positive colony bacterium order-checking in second day, comparison, completely the same with expected sequence, namely obtain a kind of expression plasmid before and after hK1 gene optimization respectively, be designated as pPIC3.5K-hK1(natural gene respectively) and pPIC3.5K-opt-hK1(gene optimization after) (plasmid construction is as shown in Fig. 6-a, 6-b).
3. simultaneously, with pUC57-opt-hK1 plasmid for template, upstream and downstream primer introduces Xho I and Not I restriction enzyme site respectively, and carry out pcr amplification, the primer sequence is as follows:
Upstream primer:
P1:GCCGCTCGAGAAGAGAGAAGCAGAGGCTATCGTC
Downstream primer:
P2:AAGGAAAAAAGCGGCCGCCTAACTATTTTCAGCGAT
Reaction cumulative volume 50 μ L, wherein concentration is that 10 μm of ol/L primers respectively add 2.5 μ L, and concentration is that the dNTP of 10mmol/L adds 1 μ L, and archaeal dna polymerase Q5 used surpasses fidelity dna polysaccharase (M0491S, purchased from New England Biolabs company), 2U/ μ L.Reaction conditions is 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 30s, and after 25 circulations, product is through 1.0% agarose gel electrophoresis analysis, and product size is consistent with expection size (740bp).The gene product DNA gel obtained is reclaimed kits.After purifying, with Xho I and Not I double digestion, be connected to pPICZ α A(V19520 with T4 ligase enzyme, purchased from Invitrogen) and pGAPZ α A plasmid (V20520, purchased from Invitrogen) in, be transformed in DH5 α competent cell, 37 DEG C of overnight incubation in the LB flat board containing Zeocin.The order-checking of second day screening positive clone bacterium, comparison, completely the same with expected sequence, the expression plasmid of the hK1 form that namely obtains recombinating, is designated as pPICZ α-opt-hK1 and pGAPZ α-opt-hK1(plasmid construction as shown in Fig. 6-c, 6-d).
embodiment 3: the different pichia spp host engineering strains of preparation containing restructuring hK1 gene
Recombinant yeast pichia pastoris can be divided into Mut+ according to its metabolizing methanol speed, Muts and Mut-tri-kinds of phenotypes.These three kinds of Methanol Utilization Phenotype (hereinafter referred to as phenotype) essence determined by recombinant vectors recombination form in pichia spp, and between them, difference is the disappearance of AOX mono-or two gene and causes the change to methyl alcohol Utilization ability height.Mut
+for methyl alcohol utilizes speed Quick-type, Muts is that methyl alcohol utilizes the slow type of speed, and Mut-is non-methanol using type.In this patent, by utilizing speed diverse ways to express restructuring hK1 to methyl alcohol.Wherein, the phenotype that the KM71 host strain containing carrier pPIC3.5K-hK1 and pPIC3.5K-opt-hK1 produces is that the methyl alcohol of Muts utilizes speed slow type recombinant yeast pichia pastoris engineering bacteria; The phenotype that X-33 host strain containing carrier pPICZ α-opt-hK1 produces is Mut
+methyl alcohol utilize speed Quick-type recombinant yeast pichia pastoris engineering bacteria; The phenotype that X-33 host strain containing carrier pGAPZ α-opt-hK1 produces is Mut
-non-methanol using type recombinant yeast pichia pastoris engineering bacteria.
MD solid medium is prepared: Invitrogen company Multi-Copy Pichia Expression Kit specification sheets provides, wherein agarose 20g/L, without amino yeast nitrogen YNB13.4g/L, and glucose 20g/L, vitamin H 4 × 10
-4g/L.
YPD solid medium is prepared: Invitrogen company Multi-Copy Pichia Expression Kit specification sheets provides, wherein yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agarose 15g/L.
1. the methyl alcohol of preparation containing codon optimized front and back restructuring hK1 gene utilizes slow type KM71 host engineering strain
According to the method for Invitrogen company Multi-Copy Pichia Expression Kit specification sheets, KM71 bacterial strain (201178, purchased from ATCC) is prepared into Electrocompetent cells.PPIC3.5K-hK1 and pPIC3.5K-opt-hK1 plasmid is contained respectively by what obtain in embodiment 2 step 2, with Sac I restriction enzyme (R0156S, purchased from New EnglandBiolabs) linearization for enzyme restriction, by linearized vector after alcohol settling, electricity transforms and enters into KM71 competence yeast cell, coat MD solid medium, cultivate until transformant is applied to containing G418(E859-5G after growing by transformant again for 30 DEG C, purchased from Amresco company) YPD solid medium, 30 DEG C cultivate until clone grow.
2. the methyl alcohol of preparation containing codon optimized rear restructuring hK1 gene utilizes Quick-type X-33 host engineering strain
By the method for step 1, X-33 bacterial strain (C18000, purchased from Invitrogen) is prepared into Electrocompetent cells.PPICZ α-opt-hK1 plasmid is contained by what obtain in embodiment 2 step 3, with the linearizing of SacI digestion with restriction enzyme, by linearized vector after alcohol settling, electricity transforms and enters into X-33 competence yeast cell, be applied to containing Zeocin(R250-01, purchased from Invitrogen company) YPD solid medium, 30 DEG C cultivate until clone grow.
3. the non-methanol using type X-33 host engineering strain of preparation containing codon optimized rear restructuring hK1 gene
By the method for step 1, X-33 bacterial strain is prepared into Electrocompetent cells.PGAPZ α-opt-hK1 plasmid is contained by what obtain in embodiment 2 step 3, with the linearizing of Sac I digestion with restriction enzyme, by linearized vector after alcohol settling, electricity transforms and enters into X-33 competence yeast cell, be applied to the YPD solid medium containing Zeocin, cultivate until clone grows for 30 DEG C.
embodiment 4: the different pichia spp host engineering strain abduction delivering containing restructuring hK1 gene and qualification
BMGY substratum is prepared: Invitrogen company Multi-Copy Pichia Expression Kit specification sheets provides, wherein yeast extract 10g/L, peptone 20g/L, K
2hPO
43g/L, KH
2pO
411.8g/L, YNB13.4g/L, vitamin H 4 × 10
-4g/L, glycerine 10g/L.
BMMY substratum is prepared: Invitrogen company Multi-Copy Pichia Expression Kit specification sheets provides, wherein yeast extract 10g/L, peptone 20g/L, K
2hPO
43g/L, KH
2pO
411.8g/L, YNB13.4g/L, vitamin H 4 × 10-
4g/L, methyl alcohol 5mL/L
1. the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes the methanol induction of slow type KM71 host engineering strain to express
KM71 positive monoclonal host strain respectively containing the restructuring hK1 gene after codon optimized in picking embodiment 3 step 1 is in 5mL BMGY substratum, and in 50mL sterile centrifugation tube 30 DEG C, 220rpm cultivates, to OD
600during=2.0-6.0, get 1mL and preserve bacterial classification, and transfer to BMMY Small Amount abduction delivering after resuspended for residue bacterium liquid, adding methyl alcohol to final concentration every 24h is 0.5%(v/v).After one week, collected by centrifugation bacterium liquid supernatant, by SDS-PAGE gel electrophoresis and Western blot analysis, (primary antibodie is Anti-Kallikrein1-Kallikrein loop, ab28289, is purchased from abcam; Two resist for Anti-Rabbit IgG – Peroxidase antibody produced in goat, and A0545-1mL, is purchased from Sigma-Aldrich) observe the brightness of expression product band.Fig. 7-a, Fig. 7-b is for utilizing slow type KM71 engineering strain abduction delivering qualification figure containing codon optimized rear restructuring hK1 gene methyl alcohol.From Fig. 7-a and Fig. 7-b, restructuring hK1 albumen utilizes in slow type KM71 engineering strain at the methyl alcohol containing codon optimized rear restructuring hK1 gene and obtains remarkable expression.
2. the methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes the abduction delivering of Quick-type X-33 host engineering strain methyl alcohol
What obtain in picking embodiment 3 step 2 utilizes Quick-type X-33 host mono-clonal engineering bacteria in 5mL BMGY substratum containing codon optimized rear restructuring hK1 gene methyl alcohol, in 50mL sterile centrifugation tube 30 DEG C, and 220rpm cultivation, to OD
600during=1.0-2.0, get 1mL and preserve bacterial classification, and transfer to BMMY Small Amount abduction delivering after resuspended for residue bacterium liquid, adding methyl alcohol to final concentration every 24h is 1% (v/v).After one week, collected by centrifugation bacterium liquid supernatant, by SDS-PAGE gel electrophoresis and Western blot analysis, observes the brightness of expression product band, Fig. 8-a, Fig. 8-b is for utilizing Quick-type X-33 engineering strain abduction delivering qualification figure containing codon optimized rear restructuring hK1 gene methyl alcohol.From Fig. 8-a and Fig. 8-b, restructuring hK1 albumen utilizes in Quick-type X-33 engineering strain at the methyl alcohol containing codon optimized rear restructuring hK1 gene and obtains remarkable expression.
3. the expression of the non-methanol using type X-33 host engineering strain containing codon optimized rear restructuring hK1 gene
Respectively the non-methanol using type X-33 host mono-clonal engineering strain containing codon optimized rear restructuring hK1 gene that obtains of picking embodiment 3 step 3 is in 5mLYPD substratum, in 50mL sterile centrifugation tube 30 DEG C, and 220rpm cultivates, to OD
600during=1.0-2.0, get 1mL and preserve bacterial classification, and bacterium liquid continuation cultivation will be remained.After one week, collected by centrifugation bacterium liquid supernatant, by SDS-PAGE gel electrophoresis and Western blot analysis, observes the brightness of expression product band, Fig. 9-a, Fig. 9-b is the non-methanol using type X-33 engineering strain abduction delivering qualification figure containing codon optimized rear restructuring hK1 gene.From Fig. 9-a and Fig. 9-b, restructuring hK1 albumen obtains remarkable expression in the non-methanol using type X-33 engineering strain containing codon optimized rear restructuring hK1 gene.In addition, compared with the pichia spp host engineering strain containing hK1 gene of recombinating with other, non-methanol using type X-33 engineering strain expression system containing codon optimized rear restructuring hK1 gene does not need methanol induction, and the restructuring hK1 albumen of expressing only has a band, illustrate that it is glycosylation modified substantially identical, albumen has higher consistence.
embodiment 5: the host's engineering strain methanol induction expression amount containing codon optimized front and back restructuring hK1 gene compares
1. compared by SDS-PAGE and do not optimize the host's engineering strain methanol induction expression amount with codon optimized rear restructuring hK1 gene containing codon
Do not optimize with KM71 host's positive monoclonal bacterial strain of codon optimized rear restructuring hK1 gene in 5mL BMGY substratum containing codon in picking embodiment 3 step 1 respectively, cultivate in 50mL sterile centrifugation tube, to OD
600during=2.0-6.0, get 1mL and preserve bacterial classification, and transfer to BMMY Small Amount abduction delivering after resuspended for residue bacterium liquid, adding methyl alcohol to final concentration every 24h is 0.5.After one week, collected by centrifugation bacterium liquid supernatant, SDS-PAGE gel electrophoresis analysis, coomassie brilliant blue staining, observe the brightness of expression product band, from Figure 10-a and Figure 10-b, the host's engineering strain methanol induction expression amount containing codon optimized rear restructuring hK1 gene is far above the host's engineering strain containing codon non-optimum combination hK1 gene.
2. compared by Bioactivity and do not optimize the host's engineering strain methanol induction expression amount with codon optimized rear restructuring hK1 gene containing codon
First according to Takashi Morita, et al.New fluorogenic substrates for α-thrombin, factor Xa, kallikreins, and urokinase, J.Biochem.82, formula in 1495-1498 (1977), preparation 50mmol/L Tris-HCl, 100mmol/L NaCl, 10mmol/L CaCl
2, pH=8.0 reaction buffer.
Then fluorogenic substrate Z-Phe-Arg-MCA is dissolved in 10mmol/L DMSO, then is diluted to 0.2mmol/L with reaction buffer.Respectively not optimizing containing codon of acquisition in embodiment 4 step 1 is added 25 μ L to 96 hole enzyme plates (237108 successively with KM71 host's engineering strain bacterium liquid supernatant nutrient solution of codon optimized restructuring hK1 gene, purchased from ThermoScientfic company) in, 2 multiple holes.Add 25 μ L0.2mmol/L fluorogenic substrate Z-Phe-Arg-MCA more respectively.After vibration mixing, at excitation wavelength 380nm, emission wavelength 460nm, reads plate once every 2min, circulates 30 times.With fluorescent signal value for ordinate zou, reading the plate time is X-coordinate, and slope is fluorescent signal value and the ratio reading the plate time, and calculate the slope in each hole, slope is higher, and the MCA that release dissociates is higher, and the relative activity of enzyme is higher, and this mono-clonal expression amount is higher.From Figure 11-a and Figure 11-b, under identical expression vector and culture condition, KM71 host's engineering strain methanol induction supernatant nutrient solution relative reactivity containing codon optimized rear restructuring hK1 gene, far above the KM71 host's engineering strain methanol induction supernatant nutrient solution containing codon non-optimum combination hK1 gene, illustrates that the expression amount of the KM71 host's engineering strain containing codon optimized rear restructuring hK1 gene by methanol induction restructuring hK1 albumen is far above the KM71 host's engineering strain containing codon non-optimum combination hK1 gene.
embodiment 6: the optimization of shake flask scale fermentation condition
1. fermention medium constituent optimization
Seed culture medium: yeast extract 10g/L, peptone 20g/L, K
2hPO
43g/L, KH
2pO
411.8g/L, YNB13.4g/L, vitamin H 4 × 10
-4g/L, glycerine 10g/L.
Basal fermentation BSM substratum: glycerine 40g/L, H
3pO
427mL/L, CaSO
42H
2o0.9g/L, K
2sO
418.2g/L, MgSO
47H
2o14.9g/L, KOH4.13g/L, 121 DEG C of sterilizing 20min.After temperature drops to 50 DEG C, use strong aqua adjust pH.
Improvement fermentation BSM substratum: glycerine 40g/L, H
3pO
49mL/L, CaSO
42H
2o0.3g/L, K
2sO
46.07g/L, MgSO
47H
2o4.97g/L, KOH1.38g/L, 121 DEG C of sterilizing 20min.After temperature drops to 50 DEG C, use strong aqua adjust pH.
Trace salts solution PMT1:CuSO
46.0g/L, KI0.8g/L, MnSO4H
2o3.0g/L, Na
2moO
42H2O0.2g/L, H
3bO
30.2g/L, CaSO
42H
2o0.5g/L, ZnCl
220g/L, FeSO
47H
2o65g/L, Biotin0.2g/L, vitriol oil 5mL/L, filtration sterilization, 2mL/L adds in fermention medium.
Obtain in picking embodiment 4 and example 5 respectively do not optimize containing codon and optimize after the recombinate different hosts engineering strain of hK1 gene be inoculated in seed culture medium, after 220rpm is cultured to 48h, by thalline 3000rpm, 10min low-temperature centrifugation, transfer to after thalline is resuspended in basal fermentation BSM substratum and improvement fermentation BSM substratum, and regulate substratum to pH=6 with strong aqua.Wherein, after not optimizing containing codon and optimizing, the methyl alcohol of restructuring hK1 gene utilizes slow type KM71 host engineering bacteria and the methyl alcohol containing codon optimized rear restructuring hK1 gene to utilize the every 24h of Quick-type X-33 host engineering bacteria to add methyl alcohol to final concentration is 1.0%(v/v), 2mL/L PMT1 Trace salts solution.After one week, carry out SDS-PAGE gel electrophoresis analysis to nutrient solution supernatant respectively.Figure 12-a, the bacterium liquid supernatant SDS-PAGE gel electrophoresis figure that Figure 12-b, Figure 12-c and Figure 12-d methyl alcohol be expressed as containing natural hK1 gene utilizes slow type KM71 host engineering bacteria, methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes slow type KM71 host engineering bacteria, methyl alcohol containing codon optimized rear restructuring hK1 gene utilizes Quick-type X-33 host engineering bacteria and the different incubation time in fermentation BSM substratum and improvement fermentation BSM substratum of the non-methanol using type X-33 host engineering bacteria containing codon optimized rear restructuring hK1.As seen from the figure, no matter whether codon optimized, restructuring hK1 host engineering strain expression amount in improvement fermentation BSM substratum of different expression vector is all obviously better than traditional zymotic basis BSM substratum, and assorted band is less, and expressing quantity reached the highest at about 7 days.
2. improvement fermentation BSM medium pH is optimized
The KM71 host's engineering strain got containing the restructuring hK1 gene after optimization in enforcement 4 step 1 is transferred in seed culture medium by 1% inoculum size, and 30 DEG C, 220rpm is cultured to 48h.By thalline 3000rpm, 10min low-temperature centrifugation, transfer to after thalline is resuspended in improvement fermentation BSM substratum, strong aqua regulates substratum to different pH, 30 DEG C, and 220rpm cultivates, and it is 1.0% (v/v) that every 24h adds methyl alcohol to final concentration.After one week, carry out SDS-PAGE gel electrophoresis analysis to induction broth, as shown in Figure 13, the optimal pH of expressing in improvement fermentation BSM substratum containing the restructuring hK1 gene KM71 host engineering strain after codon optimized is 6.0-7.0.
3. improve the optimization of abduction delivering condition in BSM substratum
Research shows that the expression of recombinant protein in pichia spp inorganic medium is by cell density, and methanol induction concentration, the impacts such as abduction delivering time, only under suitable cell density and methanol concentration, recombinant protein could realize high expression.Set up cell density and methanol induction concentration orthogonal table, by under the more different inductive condition of SDS-PAGE gel electrophoresis containing the expression amount in improvement BSM substratum of the restructuring hK1 gene KM71 host engineering strain after codon optimized.
By streak culture for the KM71 host's engineering strain containing the restructuring hK1 gene after optimization in embodiment 4 step 1, choose single bacterium colony in BMGY substratum, 30 DEG C, after 220rpm cultivates 48h, to press in table 1 15 respectively, thalline is accessed to improvement BSM substratum by 30,60,120g/L cell density, methanol induction concentration is respectively 0.5,1.0,1.5%(v/v), carry out restructuring hK1 protein expression.
Table 1. is recombinated the optimization that hK1 albumen is expressed in inorganic medium
| Cell density (g/L) | Methyl alcohol final concentration (%, v/v) | |
| 1 | 15 | 0.5 |
| 2 | 30 | 0.5 |
| 3 | 60 | 0.5 |
| 4 | 120 | 0.5 |
| 5 | 15 | 1 |
| 6 | 30 | 1 |
| 7 | 60 | 1 |
| 8 | 120 | 1 |
| 9 | 15 | 1.5 |
| 10 | 30 | 1.5 |
| 11 | 60 | 1.5 |
| 12 | 120 | 1.5 |
After inducing one week, get induction broth supernatant and carry out SDS-PAGE gel electrophoresis analysis.As shown in figure 14, the comparatively suitable expression condition of the KM71 host's engineering strain containing the restructuring hK1 gene after optimization: cell density is 30-60g/L, and methanol induction concentration is 0.5%-1.5%(v/v).Optimal expression condition is: cell density is 60g/L, and methanol induction concentration is 1.0%(v/v).
embodiment 7 is recombinated the purifying of hK1 albumen
This patent mainly adopts hydrophobic chromatography to be separated the different restructuring hK1 albumen of glycosylation modified degree, and prepackage pillar is chosen as HisTrap Phenyl HP, and concrete steps are as follows:
1. the removal of impurities pre-treatment of fermented liquid
Obtain KM71 host's engineering strain fermented liquid of the restructuring hK1 gene after containing optimization by expression method in embodiment 4 step 1,12000rpm, 15min low-temperature centrifugation collects supernatant, adds high density (NH)
2sO
4solution, makes its final concentration in supernatant be 1.0mol/L, 0.45 μm of membrane filtration.
2.HisTrap Phenyl HP hydrophobic chromatography
Use fully-automatic intelligent protein purification system (AKTA avant150, purchased from GE healcare) DOE method optimizes the restructuring hK1 fermented liquid HisTrap Phenyl HP purifying process obtained pre-treatment in UNICORN6.1 function software, finally being defined as level pad is 20mmol/L sodium phosphate, 1.0M (NH
4)
2sO
4, 10% glycerine (volume percent), pH6.0, elution buffer is 20mmol/L phosphoric acid salt, 10% glycerine (volume percent), pH6.0.(NH) is progressively successively decreased according to 0.2mol/L
2sO
4the method of concentration isocratic elution, collects each elution peak, the results are shown in Figure 15, by merging satisfactory collection tube, filtration sterilization after SDS-PAGE electrophoresis determination purity, carries out the phenetic analysis of next step restructuring hK1 albumen.Two restructuring hK1 albumen that Figure 16 shows molecular size range inconsistent are separated completely.
embodiment 8 is recombinated the phenetic analysis of hK1 albumen
1. hK1 albumen n end of recombinating measures
The mensuration of protein and peptide class drug molecule N terminal sequence is one of important step of medicine industry quality control.This test mainly adopts the N terminal Sequence Analysis based on the Edman edman degradation Edman of classics, utilize the full-automatic protein and peptide sequenator of Shimadzu (PPSQ-33A, SHIMADZU) the N terminal sequence collecting the different restructuring hK1 of the relative molecular mass that obtains after embodiment 7 purifying is analyzed, result as shown in Figure 17, N terminal amino acid residue is Ile-Val-Gly-Gly-Trp, although this shows that the restructuring hK1 albumen of Pichia anomala expression is glycosylation modified slightly different, but its N terminal sequence is identical, and consistence is higher.
2. the average molecular flow measurement of restructuring hK1 albumen
The Measuring Molecule Weight of protein and peptide class drug molecule is one of important step of medicine industry quality control.This test is by Matrix-assisted laser desorption ionization (4800Plus MALDI TOF/TOF, be purchased from AB SCIEX) the restructuring hK1 albumen relative molecular mass that the relative molecular mass collected after embodiment 5 purifying is higher and on the low side is analyzed, the results are shown in Figure 18, the higher restructuring hK1 albumen relative molecular mass size of relative molecular mass is 30928Da, and the restructuring hK1 albumen relative molecular mass size that relative molecular mass is on the low side is 28511.8Da.
3. the isoelectric point determination of restructuring hK1 albumen
The isoelectric point determination of protein and peptide class drug molecule is one of important step of medicine industry quality control.This test by multifunctional flat isoelectrofocusing system (Multiphor II, purchased from GE Healthcare) the restructuring hK1 albumen that the relative molecular mass collected after embodiment 7 purifying is higher and on the low side is analyzed, the results are shown in Figure 14, the restructuring hK1 albumen that relative molecular mass is higher and on the low side is near pI standard protein 4.45, this shows that the relative molecular mass of Pichia anomala expression restructuring hK1 albumen iso-electric point higher and on the low side is not obviously distinguished, and pI is all about 4.40.
4. restructuring hK1 albumen C.D analysis
Circular dichroism spectrum technology is a kind of fairly simple and effective technology, higher structure for the protein-based product of gene engineering expression confirms to provide means easily, measure under solution state, comparatively close to its physiological status, be the one of protein conformation in research dilute solution fast, simply, method more accurately.JASCO720(JASCO is passed through in this test) gather embodiment 7 obtain restructuring hK1 albumen absorb collection of illustrative plates at the circular dichroism (CD) of extreme ultraviolet (190-250nm).Result Figure 16 shows, and in dilute solution 10mmol/L phosphoric acid salt, the different restructuring hK1 albumen of relative molecular mass does not have notable difference at the circular dichroism spectrum of the scanning of extreme ultraviolet, illustrates that the secondary structure of the restructuring hK1 albumen of two kinds of molecular weight is basically identical.
5. the de-glycosylation analysis of restructuring hK1 albumen
Analyzed known by relative molecular mass, restructuring hK1 albumen relative molecular mass is respectively 30928Da and 28511.8Da.With N-glycosidase F, de-glycosylation is carried out to the restructuring hK1 albumen that these two kinds of different glycosylations are modified, SDS-PAGE gel electrophoresis analysis, after Figure 21 shows de-glycosylation, the restructuring hK1 albumen that molecular size range is different and E.coli express without glycosylation modified restructuring hK1(contain hK1 gene intestinal bacteria of recombinating build and the preparation of hK1 albumen of recombinating see the earlier application of applicant: 201310718972.X) molecular size range is suitable.The glycosylation modified feature that there is high mannose and modify of pichia yeast expression system, relative molecular mass (26526Da) according to escherichia coli expression hK1 purification of samples calculates known, high molecular restructuring hK1 sugar degree is 14.2%, and lower molecular weight restructuring hK1 sugar degree is 6.9%.The restructuring hK1 protein glycosylation degree of the Pichia anomala expression that molecular weight is low is low, is more suitable for for follow-up study.
The Rate activity of 6 restructuring hK1 albumen measures
6.1 fluorogenic substrate Rate activity measure
The restructuring hK1 albumen fluorogenic substrate Z-Phe-Arg7-amido-4-methylcoumarin hydrochloride different to the relative molecular mass of embodiment 7 acquisition measures its activity (see Figure 22-b).Standard substance are purchased from the pig kallikrein sterling (NO.3712030700) of thousand red pharmacy, measure its activity (see Figure 22-a) by fluorogenic substrate Z-Phe-Arg7-amido-4-methylcoumarinhydrochloride.See that the restructuring hK1 protein ratio vigor of the different relative molecular masses that Figure 22-b embodiment 7 obtains is greater than for 1000IU/mg, high molecular that different sugar modifies is described, the Bioactivity of lower molecular weight restructuring hK1 albumen do not have difference.
6.2 chromophoric substrate Rate activity measure
With reaction buffer in embodiment 5 step 2, standard substance pig kallikrein (being purchased from the red pharmacy in Changzhou thousand) is diluted 5 concentration gradient 10IU/mL, 5IU/mL, 2.5IU/mL, 1.25IU/mL, 0.625IU/mL, substrate is that D-Val-Leu-Argp-nitroanilide(is purchased from sigma-Aldrich company), be diluted to 0.2mmol/L with reaction buffer during use.Sample reaction buffer dilutes suitable multiple, in 96 orifice plates, first every hole adds the substrate 80 μ L diluted, then the standard substance diluted or each 80 μ L of each sample are added immediately respectively, Synergy H1GEN microplate reader (purchased from BioTek company) 37 DEG C, 405nm place carries out absorbance detection, 1min detects once, continuous detecting 15min.Experimental result shows, and the restructuring hK1 protein ratio vigor of the different relative molecular masses that embodiment 7 obtains is 1000IU/mg, completely the same with detected result in step 6.1.Further illustrate high molecular that different sugar modifies, the Bioactivity of lower molecular weight restructuring hK1 albumen do not have difference.
In sum, through the restructuring hK1 albumen that the molecular mass of Yeast engineering bacteria expression is different, although glycosylation modified slightly different and cause molecular weight slightly difference, the two does not have difference in properties such as protein amino acid sequence, iso-electric point, space structure and biological activitys.
Claims (10)
1. an encoding gene for recombinant human kallikrein 1, is characterized in that, its base sequence is as shown in SEQ ID NO:1.
2. encoding gene as claimed in claim 1, is characterized in that, also add just like secreting signal peptide shown in SEQ ID NO:3 before its base sequence.
3. recombinate the expression method of hK1 albumen, described method comprises following step:
A. the carrier containing, for example encoding gene described in claim 1 or 2 is built;
B. proceed to after vector linearization in Pichi strain, and cultivate under suitable conditions;
C. protein purification is reclaimed.
4. method as claimed in claim 3, it is characterized in that, described carrier is pPIC3.5K, pPICZ α A or pGAPZ α A, and/or described bacterial strain is KM71 or X-33 bacterial strain.
5. method as claimed in claim 3, is characterized in that: the carrier described in steps A is pGAPZ α A, and Pichi strain described in step B is X-33 bacterial strain.
6. method as claimed in claim 3, it is characterized in that: when expressing with methanol induction, cell density is 30-60g/L, and methanol induction concentration is 0.5%-1.5%.
7. ferment a BSM substratum, contains: glycerine 40g/L, H
3pO
49mL/L, CaSO
42H
2o0.3g/L, K
2sO
46.07g/L, MgSO
47H
2o4.97g/L, KOH1.38g/L, and the Trace salts solution PMT1 of 2mL/L;
Described Trace salts solution PMT1 contains: CuSO
46.0g/L, KI0.8g/L, MnSO
4h
2o3.0g/L, Na
2moO
42H
2o0.2g/L, H
3bO
30.2g/L, CaSO
42H
2o0.5g/L, ZnCl
220g/L, FeSO
47H
2o65g/L, Biotin0.2g/L, vitriol oil 5mL/L;
As preferably, described fermentation BSM substratum strong aqua regulates pH to be 6.0-7.0.
8. a tissue kallikrein protein enzyme kinetics activity test method, described method comprises following step:
A. fluorogenic substrate Z-Phe-Arg-MCA is dissolved in DMSO;
B. added in enzyme plate by the substratum containing tissue kallikrein protein, then add fluorogenic substrate Z-Phe-Arg-MCA respectively, after vibration mixing, arranging excitation wavelength is 360-400nm, and emission wavelength is 440-480nm, and plate is read in circulation;
C. with fluorescent signal value for ordinate zou, reading the plate time is X-coordinate, and slope is fluorescent signal value and reads the ratio of plate time, and calculate the slope in each hole, this protein-active of the larger expression of slope is higher.
9. the application of detection method as claimed in claim 8 in the screening of high expression level monoclonal cell strain.
10. an albumen hydrophobic chromatography purification process for recombinant tissue kallikrein 1, described purification process is as follows:
A. by recombinant tissue kallikrein 1 fermented liquid low-temperature and high-speed collected by centrifugation supernatant, high density (NH) is added
2sO
4solution, makes its final concentration in supernatant be 1.0mol/L, membrane filtration;
B. first use equilibration buffer pillar, the restructuring hK1 fermented liquid then pre-treatment in steps A obtained, by prepacked column or separating filler, then uses elution buffer progressively to successively decrease (NH) according to 0.2mol/L
2sO
4concentration isocratic elution, collects each elution peak, namely obtains the restructuring hK1 albumen that glycosylation modified degree is different;
Described level pad is preferably containing 20mmol/L sodium phosphate, 1.0mol/L (NH)
2sO
4, 10% glycerine (volume percent), pH6.0; Described elution buffer is preferably containing 20mmol/L phosphoric acid salt, 10% glycerine (volume percent), pH6.0.
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| CN106520796A (en) * | 2016-11-04 | 2017-03-22 | 江苏众红生物工程创药研究院有限公司 | Recombinant N-PNGase F and encoding gene and application thereof |
| CN106929496A (en) * | 2015-12-31 | 2017-07-07 | 江苏众红生物工程创药研究院有限公司 | A kind of pharmaceutical grade recombined human kininogenase industrialization production method |
| CN113073092A (en) * | 2021-04-15 | 2021-07-06 | 宁波瑞林生物科技有限公司 | Recombinant human tissue kallikrein and preparation method thereof |
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| CN101395269A (en) * | 2002-10-10 | 2009-03-25 | 戴弗萨公司 | Proteases, nucleic acids encoding them and methods for making and using them |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106929496A (en) * | 2015-12-31 | 2017-07-07 | 江苏众红生物工程创药研究院有限公司 | A kind of pharmaceutical grade recombined human kininogenase industrialization production method |
| CN106929496B (en) * | 2015-12-31 | 2020-05-12 | 江苏众红生物工程创药研究院有限公司 | Industrialized production method of pharmaceutical-grade recombinant human kininogenase |
| CN106520796A (en) * | 2016-11-04 | 2017-03-22 | 江苏众红生物工程创药研究院有限公司 | Recombinant N-PNGase F and encoding gene and application thereof |
| CN113073092A (en) * | 2021-04-15 | 2021-07-06 | 宁波瑞林生物科技有限公司 | Recombinant human tissue kallikrein and preparation method thereof |
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