CN1214107C - Enterpeptidase light chain variant with high activity and high stability - Google Patents
Enterpeptidase light chain variant with high activity and high stability Download PDFInfo
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- CN1214107C CN1214107C CN 02138017 CN02138017A CN1214107C CN 1214107 C CN1214107 C CN 1214107C CN 02138017 CN02138017 CN 02138017 CN 02138017 A CN02138017 A CN 02138017A CN 1214107 C CN1214107 C CN 1214107C
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- light chain
- enterpeptidase light
- enterpeptidase
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Abstract
The site-specific mutagenesis is carried out on 2-amino-3-thiopropionic acid in the 112th site of an enterokinase light chain gene. An enterokinase light chain variant is expressed by colibacillus and yeast, and carries out Zn-Sepharose and STI-Sepharose affinity chromatograph, and then high-purity enterokinase light chain variant protein is prepared. Compared with the wild enterokinase light chain, the variant has high stability and enzyme activity.
Description
Technical field
The present invention relates to a kind of light chain variant of serine proteinase enzyme, particularly enteropeptidase.
Background technology
(Enterokinase EK) is one of most basic serine proteinase enzyme in the Mammals Digestive tract to enteropeptidase.Under physiological condition, the EK that is anchored on the cytolemma is trypsinase with the trypsinogen activation, and trypsinase activates other proenzymes in the pancreas again and then forms a series of proteolytic enzyme mixt.
Enteropeptidase is made up of two polypeptide chains, i.e. heavy chain and light chain, and connect by a pair of interchain disulfide bond, the wherein Cys in the light chain
112Participated in and being connected of heavy chain.The light chain of enteropeptidase has complete catalysis, its recognition sequence be Asp-Asp-Asp-Asp-Lys ↓.Enteropeptidase has the specificity of height to its recognition sequence, is widely used in the peptide bond between the carrier proteins and target protein in the cleavage of fusion proteins at present, because P
1The amino-acid residue in ' site is very little to the specificity and the activity influence of enteropeptidase.In addition, fusion rotein has and the on all four aminoacid sequence of wild-type through the target protein that the EK enzymolysis obtains.
But at present commercially available enteropeptidase price is very expensive, wherein often there is the pollution of other protease in the enteropeptidase of separation and purification from tissue, and wild-type Enterpeptidase light chain (EKLC) less stable by genetically engineered preparation, the long-time preservation is easy to generate polymer or precipitation, causes enzymic activity to descend.We think the free Cys that exists in the Enterpeptidase light chain molecule
112Can cause the intermolecular polymerization of Enterpeptidase light chain, and cause the reduction of enzymic activity.
Summary of the invention
The objective of the invention is to utilize genetic engineering technique, clone's Enterpeptidase light chain gene and to Cys
112Codon carries out rite-directed mutagenesis, is respectively charged into protokaryon or eukaryotic expression vector again, and adopts the mode of protokaryon or eukaryotic cell fermentation to obtain stability and the high Enterpeptidase light chain variant albumen of enzymic activity.
Technical solution of the present invention:
A kind of Enterpeptidase light chain variant with high reactivity and high stability, it is characterized in that by gene engineering method, to the cysteine residues rite-directed mutagenesis of the 112nd in Enterpeptidase light chain albumen with protokaryon or eukaryotic cell preparation; Do not have the free cysteine residues in the final organized enzyme product, and make the stability and active raising of enzyme.
Advantage of the present invention
1. adopt the Zn-Sepharose affinity chromatography to carry out purifying to the expression product of Enterpeptidase light chain variant in protokaryon or eukaryotic cell, method is simple, and cost is low.
2. when adopting prokaryotic cell prokaryocyte to prepare Enterpeptidase light chain or its variant, the activity of Enterpeptidase light chain variant reclaims and is higher than the corresponding wild-type Enterpeptidase light chain.
Zhi Bei Enterpeptidase light chain variant to the hydrolytic activity of small molecules substrate a little more than the wild-type Enterpeptidase light chain, and has higher zymetology stability, show as with fusion rotein be incubated for a long time the back (＞10hr), the Enterpeptidase light chain variant of identical activity unit is significantly higher than the corresponding wild-type Enterpeptidase light chain to the enzymolysis of fusion rotein.
Description of drawings
Fig. 1 is from the wild-type Enterpeptidase light chain of yeast preparation and the Enterpeptidase light chain variant electrophoretogram to the enzymolysis of thioredoxin-human brain natriuretic peptide fusion rotein.
Fig. 2 is from the wild-type Enterpeptidase light chain of intestinal bacteria preparations and the Enterpeptidase light chain variant electrophoretogram to the enzymolysis of thioredoxin-human brain natriuretic peptide fusion rotein.
Fig. 3 forms dimeric electrophoretogram with wild-type Enterpeptidase light chain and variant thereof that yeast prepares.
Embodiment
The present invention realizes in the following manner:
1. fresh ox or people's duodenum tissue is got in the preparation of the total RNA of duodenum, extracts total RNA according to Qigen kit (Catalog NO.74104) operational manual.
2.RT-PCR reaction
The RT-PCR test kit is available from Invitrogen company, and (Catalog No.L1310-01) experimentation is with reference to operational manual.Get the total RNA of 3 μ g, add water to final volume 11.5 μ l, add 1 μ l thymus pyrimidine Oligonucleolide primers again, mixing, put 65 ℃ of insulation 10min, room temperature 2min adds 1 μ l RNase inhibitor more successively, 4 μ l 5x reaction buffers, 1 μ l 100mM dNTP, 1 μ l 80mM trisodium phosphate, 0.5 μ l AMV ThermoScript II, mixing is put 42 ℃ of reaction 60min.
Get the above-mentioned reaction solution of 3 μ l as the pcr amplification template.When adopting prokaryotic cell prokaryocyte as the expressive host bacterium, 5 of pcr amplification ' end primer is
CAT ATG GAC GAC GAT GAC AAG ATT GTC GGA GGA AGT GAC TCC, 3 ' end primer is
CTC GAG ATG TAG AAA ACT TTG TAT CCA CTCWhen adopting yeast as the expressive host bacterium, 5 of pcr amplification ' end primer is
CTC GAG AAA AGA ATT GTC GGA
30 circulations
GGA AGT GAC TCC, 3 ' end primer is
GTC GAC ATG TAG AAA ACT TTG TAT CCA CTC(annotate in 3 ' end primer and all do not contain terminator codon, but 6 poly Histidines on the employing expression vector and termination codon thereof), add 50pmol 5 ' end and 3 ' hold primer, the PCR damping fluid of 5 μ l 10x more respectively, 5 μ l 10mM dNTP and 0.25U Taq enzyme, and use H
2The O constant volume is to 50 μ l.The condition of pcr amplification is:
3. the structure of Enterpeptidase light chain gene clone plasmid (PCR2.1EKLC)
Pcr amplification product is directly connected to (Invitrogen company product, Catalog No.K2040-40) on the PCR2.1 carrier.Get 3 μ l pcr amplification products, add 1 μ l PCR2.1 carrier, 1 μ l salts solution, 1 μ l water is put room temperature and is connected 4min.Get 3 μ l and connect product conversion TOPO-10 competent cell.Containing picking colony on the LB agar plate of penbritin and kantlex, cut screening positive clone with the EcoRI enzyme, the step of going forward side by side is carried out Sequence Identification.
4. Enterpeptidase light chain Cys
112Rite-directed mutagenesis (PCR2.1EKLC/Cys
112→ Ala)
Adopt the rite-directed mutagenesis test kit of Stratagene company to carry out Cys
112Rite-directed mutagenesis (Catalog No.200518).Mutant primer is:
①GAT?TAC?ATA?CAA?CCT?ATT?GCT?TTA?CCG?GAA?GAA?AATCAA
②TTG?ATT?TTC?TTC?CGG?TAA?AGC?AAT?AGG?TTG?TAT?GTAATC
Get PCR 2.1EKLC plasmid 200ng, add the amplification buffer of 50pmol primer 1 and 2,5 μ l10x respectively, 5 μ l 25mM dNTP, and the Pfu enzyme of 0.5u, and the water constant volume is to 50 μ l.
Amplification condition is:
95 ℃---→ 90 seconds
| 95 ℃ 50 ℃ 68 ℃----→----→--30 seconds 60 |
18 circulations
Amplification finishes, and adds 1 μ l DpnI and puts 37 ℃ of reactions 1 hour.Get the above-mentioned reaction soln of 3 μ l and transform the JM109 competent cell.Contain picking colony on the LB agar plate of penbritin and kantlex, and carrying out the sequencing checking.
When 112 cysteine residues to the enteropeptidase protein sequence carry out point mutation, or keep the wild-type in other sites of its gene order or protein sequence, or simultaneously other single sites of the gene order of enteropeptidase or protein sequence or multidigit point are suddenlyd change or lack or insert, can both reach effect of the present invention.
5. the structure of Enterpeptidase light chain variant expression vector
When making up the procaryotic cell expression plasmid, with PCR2.1EKLC/Cys
112→ Ala plasmid and expression vector PET29b NdeI/XhoI double digestion.PCR2.1EKLC/Cys
112→ Ala plasmid enzyme restriction product reclaims the purifying small segment after 1% agarose gel electrophoresis separation.The PET29b enzyme cut product then purifying reclaim big fragment.Get each 8.5 μ l of above-mentioned two fragments, add the connection damping fluid of 2 μ l10x and the ligase enzyme of 1 μ l, put 15 ℃ of connections and spend the night.Get 5 μ l and connect product conversion TOPO-10 competent cell.Containing picking colony on the agar plate of kantlex, and cutting screening positive clone (PET29bEKLC/Cys112 → Ala) with the NdeI/XhoI enzyme.
When making up yeast expression system, with PCR2.1EKLC/Cys
112→ Ala plasmid and expression vector pPICZ α A XhoI/AccI double digestion, after agarose gel electrophoresis separates, PCR2.1EKLC/Cys
112→ Ala plasmid enzyme restriction product purification reclaims small segment, and pPICZ α A enzyme is cut product purification and reclaimed big fragment.Respectively get the above-mentioned fragment of 8.5 μ l and mix, add 2 μ l and connect damping fluid and 1 μ l ligase enzyme, put 15 ℃ of connections and spend the night, get 5 μ l and connect product conversion TOPO-10 competent cell.Containing picking colony on the less salt LB agar plate of Zeocin, cutting screening positive clone (pPICZ α A EKLC/Cys112 → Ala) with the XhoI/AccI enzyme.
6. the foundation of engineering bacteria
1. with intestinal bacteria the host bacterium
With Enterpeptidase light chain variant gene (PET29bEKLC/Cys
112→ Ala) transform BL21, containing picking colony on the LB agar plate of kantlex, the reduction SDS-PAGE with 12% analyzes and compares each bacterial strain through IPTG inductive expression efficiency, and the bacterial strain of screening expression efficiency the highest (15% ± 2%) is as engineering bacteria.
2. with the yeast host bacterium
With SacI single endonuclease digestion Enterpeptidase light chain variant gene (pPICZ α AEKLC/Cys
112→ Ala), make plasmid linearization, according to the Invitrogen operational manual, above-mentioned linearizing plasmid is transformed GS115 finish red (pichia) competent cell.Containing picking list bacterium colony on the YPDS agar plate of Zeocin, be inoculated in 25ml BMGY substratum, 30 ℃ with 280rpm rotating speed jolting 18 hours, make saccharomycetic nectar degree OD
600About=5.Room temperature centrifugal 5 minutes with 3000g, remove supernatant, with BMMY substratum dilution thalline to OD
600=1, added methyl alcohol to final concentration every 24 hours and be 0.5% to induce the secreting, expressing of Enterpeptidase light chain, relatively the expression efficiency of each bacterial strain.
7. the preparation of Enterpeptidase light chain variant
1. intestinal bacteria are as the expressive host bacterium
1) engineering bacteria centrifugal collection thalline after IPTG induces, ratio in every gram thalline 5ml adds lysis buffer (25mM Tris-HCl, 6M Guanidinium hydrochloride, 10mM beta-mercaptoethanol, pH7.9), carrying out ultrasonic bacteria breaking, the centrifuging and taking supernatant liquor, last sample is to the Zn-Sepharose affinity column, with lavation buffer solution (25mM Tris-HCl, 8M urea, 20mM imidazoles, 0.5M NaCl, the 10mM beta-mercaptoethanol, pH7.9) flush away impurity composition is with elution buffer (25mMTris-HCl, 8M urea, the 200mM imidazoles, 0.3M NaCl, 10mM-β mercaptoethanol, pH7.9) wash-out Enterpeptidase light chain variant.
2) with the Enterpeptidase light chain sample renaturation buffer (25mM Tris-HCl, 2M urea, the 2mM CaCl that collect
2, the 1.5mM Sleep-promoting factor B, the 0.75mM reduced glutathion, pH8.0) dilution is 25 times, after room temperature is placed 18 hours, to 25mM Tris-HCl, 2mM CaCl
2, the damping fluid of pH7.5 is fully dialysed, the centrifuging and taking supernatant liquor.
3) with sample on the supernatant liquor to the STI-Sepharose post, with lavation buffer solution (25mMTris-HCl, 0.5M NaCl, pH7.5) thorough washing is with elution buffer (25mM sodium formiate, 0.3M NaCl, pH3.0) with the Enterpeptidase light chain variant wash-out, elution peak is to 25mMTris-HCl, and pH7.5 fully dialyses, and is standby.
2. use yeast as the expressive host bacterium
With methanol induction Enterpeptidase light chain secreting, expressing after 72 hours, collected supernatant liquor at 4 ℃ in centrifugal 15 minutes with 7500rpm, 25mM Tris-HCl pH7.9 damping fluid is fully dialysed and gone up sample to the Zn-Sepharose post, lavation buffer solution (25mMTris-HCl with 10 times of bed volumes, the 20mM imidazoles, 0.5M NaCl, pH7.9) thorough washing, with elution buffer (25mM Tris-HCl, the 200mM imidazoles, 0.3M NaCl is pH7.9) with Enterpeptidase light chain or its misfolded proteins wash-out from the Zn-Sepharose post.
8. the property analysis of Enterpeptidase light chain variant
1. (enzymatic reaction kinetics of Gly-Asp-Asp-Asp-Lys-β-naphthylamide) is measured to the small molecules substrate
Enzymatic reaction kinetics is measured the method with reference to Grant and Hermon-Taylor
[1], measurement result: from its Km of Enterpeptidase light chain of intestinal bacteria preparation is 0.52mM, and kcat is 37.2S
-1Its Km of Enterpeptidase light chain variant is 0.58mM, and kcat is 46.5S
-1From its Km of Enterpeptidase light chain of yeast preparation is 0.76mM, and kcat is 59.4S
-1Enterpeptidase light chain variant Km is 0.81mM, and kcat is 68.3S
-1The above results show Enterpeptidase light chain variant to the hydrolytic activity of small molecules substrate a little more than the corresponding wild-type Enterpeptidase light chain.
2. to the enzymolysis of thioredoxin-B-Type natriuretic peptide fusion rotein
(20mM Tris-HCl, 2mM CaCl in 200 μ l enzymolysis damping fluids
20.1M NaCl, pH7.5) add Enterpeptidase light chain albumen or the Enterpeptidase light chain variant of 400 μ g thioredoxin-B-Type natriuretic peptide fusion roteins and 8u respectively, put 37 ℃ of insulations after 6～18 hours, get 30 μ l samples, the Tricine SDS-PAGE with 16% detects the enzymolysis degree of fusion rotein.
Fig. 1 has shown from the wild-type Enterpeptidase light chain (EKLC/Cys of yeast preparation
112) and Enterpeptidase light chain variant (EKLC/Cys
112→ Ala) to the enzymolysis of thioredoxin-human brain natriuretic peptide fusion rotein (Trx-BNP).Trx and BNP represent thioredoxin and human brain natriuretic peptide respectively among the figure.The 5th swimming lane is the Trx-BNP contrast; The wild-type Enterpeptidase light chain that the 2nd, 4,7 swimming lanes are respectively 8u was 37 ℃ of enzymolysis Trx- BNP 12,6,16 hours; The Enterpeptidase light chain variant that the 1st, 3,6 swimming lanes are respectively 8u was 37 ℃ of enzymolysis Trx- BNP 12,6,16 hours.This presentation of results is higher than the wild-type Enterpeptidase light chain with the Enterpeptidase light chain variant of yeast preparation to the enzymolysis activity of Trx-BNP fusion rotein.
Fig. 2 has shown from the wild-type Enterpeptidase light chain (EKLC/Cys of intestinal bacteria preparation
112) and Enterpeptidase light chain variant (EKLC/Cys
112→ Ala) to the enzymolysis of Trx-BNP fusion rotein.Wherein the 1st, 3, the 5 swimming lanes wild-type Enterpeptidase light chain that is respectively 8u was 37 ℃ of enzymolysis Trx- BNP 18,6,12 hours; The 2nd, 4,6 swimming lanes are respectively Enterpeptidase light chain variant 37 ℃ of enzymolysis Trx- BNP 18,6,12 hours.This presentation of results is from the Enterpeptidase light chain variant (EKLC/Cys of intestinal bacteria preparation
112→ Ala) enzymolysis activity to fusion rotein is higher than wild-type Enterpeptidase light chain (EKLC/Cys
112).
3. the stability of Enterpeptidase light chain and misfolded proteins thereof
Use 20mM Tris-HCl, 0.1M NaCl, 5mM EDTA, 100KIU/ml Aprotinin, Enterpeptidase light chain and the misfolded proteins thereof of pH7.5 damping fluid preparation 0.4mg/ml, after putting 37 ℃ of insulations 24 hours or putting room temperature 72hr, the non-reduced SDS-PAGE with 12% examines and determine the polymer content of above-mentioned sample.
Fig. 3 has shown the wild-type Enterpeptidase light chain (EKLC/Cys with the yeast preparation
112) and variant (EKLC/Cys
112→ Ala) form dimeric effect.The monomeric molecular weight of Enterpeptidase light chain is 34K dalton among the figure, and Enterpeptidase light chain dimer molecule amount is 68K dalton.The 1st swimming lane be molecular weight standard (from top to bottom, the molecular weight of 5 bands is respectively 97Kd, 66Kd, 43Kd, 31Kd, 14.4Kd); The 2nd, 3 swimming lane is respectively Enterpeptidase light chain variant and wild-type Enterpeptidase light chain; The 4th, 5 swimming lane is respectively Enterpeptidase light chain and the wild-type Enterpeptidase light chain is incubated 24 hours at 37 ℃; The 6th, 7 swimming lane is respectively Enterpeptidase light chain variant and the wild-type Enterpeptidase light chain was placed 3 days in room temperature.The result shows that the wild-type Enterpeptidase light chain produces dimer in room temperature or 37 ℃ of meetings of placement, and Enterpeptidase light chain variant is not observed polymeric formation under similarity condition, illustrate that Enterpeptidase light chain variant has higher stability than its wild-type.
Embodiment one
Adopt the BL21 engineering bacterium fermentation of Enterpeptidase light chain variant.With 10 liters of fermentor tanks is example (fermentation time continues 2 days), every liter of fermented liquid can get thalline 27.8 grams, contain the about 578mg of Enterpeptidase light chain variant albumen, obtain the Enterpeptidase light chain variant albumen of 415mg sex change through the Zn-Sepharose affinity chromatography, handle every liter through renaturation and obtain enterokinase activity 2.19 * 10
5U through the STI-Sepharose affinity chromatography, reclaims enterokinase activity 1.64 * 10
5U, per 10 liters of fermented liquids obtain 1.64 * 10
6The u enterokinase activity.Calculate the fusion rotein of enteropeptidase degradable 82 grams that per 10 liters of fermented liquids obtain by each the enterokinase activity hydrolysis 50ug of unit fusion rotein.
Adopt the Enterpeptidase light chain albumen of method for preparing wild-type, per 10 liters of fermented liquids only obtain 1.18 * 10
6The enterokinase activity of u.This is because the annealing efficiency of Enterpeptidase light chain variant is high by 32 ± 5% than wild-type.
Embodiment two
Adopt the Yeast engineering bacteria fermentation (fermentation time continues 96 hours) of Enterpeptidase light chain variant.With 10 liters of fermentor tanks is example, and the enterokinase activity that contains in every liter of fermented liquid supernatant is 1.45 * 10
6U after the Zn-Sepharose affinity chromatography, reclaims enterokinase activity 1.03 * 10
6U, 10 liters of fermented liquids obtain 1.03 * 10
7The u enterokinase activity calculates by each unit enterokinase activity hydrolysis 50 μ g fusion rotein, 10 liters of enteropeptidase degradable 515 gram fusion roteins that fermented liquid obtains.
Reference:
Grant，D.A.W.&?Hermon-Taylar，J.Hydrolysis?of?artificial?substrates?byenterokinase?and?trypsin?and?the?development?of?a?sensitive?specific?assayfor?enterokinase?in?serum.Biochim.Biophys.Acta，(1979)；567，207-215.
Claims (1)
1. Enterpeptidase light chain variant that obtains by gene engineering method with high reactivity and high stability, the amino acid that it is characterized in that the 112nd in Enterpeptidase light chain albumen is Ala by the Cys rite-directed mutagenesis of wild-type, and other site of protein sequence still is left the site of wild-type.
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| CN100557019C (en) * | 2007-05-11 | 2009-11-04 | 上海张江生物技术有限公司 | Recombination ox intestine kinase, Preparation Method And The Use |
| EP2794875B1 (en) * | 2011-12-23 | 2020-07-22 | Novo Nordisk A/S | Modified enterokinase light chain |
| CN103998606B (en) * | 2011-12-23 | 2018-04-17 | 诺沃—诺迪斯克有限公司 | The Enterpeptidase light chain of modification |
| CN104911166A (en) * | 2014-12-15 | 2015-09-16 | 上海张江生物技术有限公司 | Novel recombinant medaka enteropeptidase light chain protein |
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