CN101864407A - TEV protease mutant, coding gene and application thereof - Google Patents
TEV protease mutant, coding gene and application thereof Download PDFInfo
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Abstract
本发明涉及一种TEV蛋白酶突变体，与原始TEV蛋白酶相比，第17位由丝氨酸(Ser)取代苏氨酸(Thr)，第56位由缬氨酸(Val)取代亮氨酸(Leu)，第68位由天冬氨酸(Asp)取代天冬酰胺(Asn)，第77位由缬氨酸(Val)取代异亮氨酸(Ile)，第135位由甘氨酸(Gly)取代丝氨酸(Ser)。本发明还提供了突变体的编码基因及其含有编码基因的表达载体和宿主细胞以及突变体在大肠杆菌中进行蛋白重组表达的应用。采用定点突变技术改造TEV蛋白酶基因，特异性的改变TEV蛋白酶中的五个氨基酸残基，获得的TEV蛋白酶的突变体可溶性和酶活性均得到显著的提高。
The present invention relates to a TEV protease mutant. Compared with the original TEV protease, the 17th position is substituted by serine (Ser) for threonine (Thr), and the 56th position is substituted by valine (Val) for leucine (Leu). , the 68th position is replaced by aspartic acid (Asp) asparagine (Asn), the 77th position is replaced by valine (Val) isoleucine (Ile), and the 135th position is replaced by glycine (Gly) Serine ( Ser). The invention also provides the coding gene of the mutant, the expression vector containing the coding gene and the host cell, and the application of the mutant in Escherichia coli for protein recombinant expression. The site-directed mutagenesis technique is used to transform the TEV protease gene, specifically changing five amino acid residues in the TEV protease, and the obtained TEV protease mutant has significantly improved solubility and enzyme activity.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to grow tobacco plaque virus (TEV) protease mutant, the encoding gene of this mutant and a method for preparing this mutant.
Background technology
In intestinal bacteria, carry out albumen when recombinant expressed, a lot of protein be not expression amount not the higher position be that solubility is very low.Make the expression amount of target protein rise or the solubility raising, also target protein and some specific albumen label amalgamation and expressions can be transformed target protein on gene level, produce the recombinant protein that contains the albumen label.The albumen label of using at present has a lot, comprise maltose binding protein commonly used (maltose-binding protein, MBP), Thiadiazolidine isomerase (glutathione S-transferase, GST), and Trx (thioredoxin, Trx), little ubiquitin relevant modifications thing (small ubiquitin-related modifier, SUMO) etc.These albumen labels can make the Recombinant Protein Expression amount rise, and solubility significantly improves, for expression, the purifying of target protein are provided convenience.Yet these fusion tags at N-end or C-end may be to the structure and the certain influence of active generation of target protein.Therefore structure or the function at research protein or enzyme needs these labels are removed after purifying is finished often.
Though chemistry and Enzymology method can both excise the albumen label,, the chemical method specificity is not high, and the cutting condition is gentle inadequately, usually can cause the irreversible inactivation of target protein.Have only the use Enzymology method, just can reach higher specificity, therefore excising albumen label method commonly used is to introduce one section proteolytic enzyme enzyme to cut sequence between label and target protein, uses special proteolytic enzyme that the albumen label is excised.The proteolytic enzyme of in the past selecting for use mainly comprises zymoplasm (Thrombin), factor Xa (Factor Xa), enteropeptidase (Enterokinase) etc., when wherein factor Xa and enteropeptidase cut, can not stay unnecessary amino-acid residue, help the research of native protein 26S Proteasome Structure and Function at the N-of target protein end.But all to exist restriction enzyme site not single-minded for these proteolytic enzyme according to the literature, and enzyme cuts often that regular meeting causes shortcomings such as target protein degraded.And TEV proteolytic enzyme obtains people and more and more payes attention to owing to the recognition sequence specificity of its height.
TEV proteolytic enzyme (tobacco etch virus protease) is the catalysis region of 27kD in the tobacco plaque virus N Ia albumen, recognition protein preface Glu-Xaa-Xaa-Tyr-Xaa-Gln-Gly/Ser (the most frequently used sequence is Glu-Asn-Leu-Tyr-Phe-Gln-Gly) specifically, and in the end cut between two amino-acid residue Gln (P1) and the Gly (P1 '), its sequence specificity is more than proteolytic enzyme height such as Thrombin, Factor Xa, Enterokinase.After though TEV albumen cuts target protein, can stay next amino-acid residue at the N-of target protein end, but the P1 ' position of restriction enzyme site, be not narrow spectrum Gly of being or Ser, P1 ' position is except that Pro influences the cutting effect of TEV proteolytic enzyme very significantly, other amino acid can obviously not influence the cutting of the right fusion rotein of TEV proteolytic enzyme, by prolonging the concentration of clipping time or increase TEV proteolytic enzyme, can make that all most fusion rotein is cut.Therefore, can after cutting, enzyme obtain the natural amino acid that the N-end does not contain unnecessary amino-acid residue by design.Simultaneously, TEV proteolytic enzyme can tolerate multiple common proteinase inhibitor, even and the cutting efficiency of TEV proteolytic enzyme also higher in the time of 4 ℃, so TEV proteolytic enzyme is widely used in protein research.
But there are certain defective in the expression and the purifying of TEV proteolytic enzyme self.At first, intramolecularly can take place from hydrolysis in natural TEV proteolytic enzyme, in the process of expression and purifying natural TEV proteolytic enzyme, it can constantly occurred conformation changes owing to the collision of other TEV proteolytic enzyme, the specific site of himself is cut, thereby make complete proteolytic enzyme by brachymemma, activity reduces greatly.Biochemical and structure studies show that this specific cleavage site is quite near from enzyme active center.For overcoming this difficulty, people such as Lucast have obtained the S219N mutant, and its stability is greatly improved, but the S219N mutant is the same with original TEV proteolytic enzyme when expressing, solubility is not high, and it is that form with inclusion body is present in the precipitation that about 95% albumen is arranged.The gene order of natural TEV proteolytic enzyme that people such as Kapust have used the genetically engineered point mutation, obtaining the higher and enzymic activity of this stability of S219V also has the mutant that improves a little, and its stability will exceed about 100 times than S219N.
Secondly, it is not high that TEV proteolytic enzyme is expressed output, and solubleness is also very low.Approximately have only 5% TEV proteolytic enzyme to be present in the supernatant of cytoclasis liquid, output is 12.5mg/L.People such as Van denBerg have found a kind of mutant TEV that output can be brought up to 54mg/L by gene reorganization (DNA shuffling), fallibility PCR (error-prone PCR)
SH, its solubleness improves a lot than S219N, but still has a large amount of TEV proteolytic enzyme to be present in the precipitation with insoluble form, and enzymic activity changes little.People such as Lei Fang are cloned into two different carriers respectively with TEV proteolytic enzyme and molecular chaperones, carry out coexpression at low temperatures, make TEV proteolytic enzyme output bring up to 65mg/L, and enzymic activity obtain reservation to a certain degree.Cabrita, L.D. wait the people to use the PoPMuSiC software design, TEV proteolytic enzyme simple point mutation body is carried out stability analysis, five mutant have been filtered out, their solubility and enzymic activity all get a promotion with respect to wild-type TEV proteolytic enzyme, also obtain a double-mutant simultaneously, its solubility and enzymic activity are significantly increased with respect to single mutant.Blommel, people such as P.G are with TEV proteolytic enzyme and maltose binding protein (MBP) amalgamation and expression, and people such as Wu Xudong are with TEV proteolytic enzyme and super folding green fluorescent protein (sfGFP) amalgamation and expression, and the Expression of Fusion Protein amount all is significantly improved.People such as Kraft are by fluorogenic substrate, develop a cover can practical method, it can detect the expression in vivo of TEV proteolytic enzyme effectively with folding, for the further research of TEV proteolytic enzyme provides a kind of method efficiently and effectively.
Summary of the invention
The present invention is directed to above-mentioned TEV proteolytic enzyme self deficiency, a kind of solubility and the enzymic activity mutant that all more original TEV proteolytic enzyme is significantly improved is provided, and the method for the encoding gene of this mutant and this mutant of preparation.
The present invention can cross following technical scheme to realize:
First purpose of the present invention provides a kind of TEV protease mutant, compare with original TEV proteolytic enzyme, the 17th by Serine (Ser) replacement Threonine (Thr), the 56th by Xie Ansuan (Val) replacement leucine (Leu), the 68th by aspartic acid (Asp) replacement l-asparagine (Asn), the 77th replaces Isoleucine (Ile) by Xie Ansuan (Val), and the 135th replaces Serine (Ser) by glycine (Gly).
Described TEV protease mutant has the aminoacid sequence shown in the sequence 2 in the sequence table.
Second purpose of the present invention provides the gene of coding TEV protease mutant, and its nucleotide sequence is shown in sequence in the sequence table 1.
The 3rd purpose of the present invention provides a kind of aforementioned expression carrier pET28b that contains.
The 4th purpose of the present invention provides a kind of host cell intestinal bacteria Rosetta (DE3) competence that contains aforementioned gene and aforementioned bearer.
The present invention's the 5th purpose is that the TEV protease mutant carries out the recombinant expressed application of albumen in intestinal bacteria.
Preparation principle of the present invention is: external rite-directed mutagenesis (site-directed mutagenesis) technology is the strong instrument of the complex relationship between research protein structure and the function, also be transformation/optimized gene means commonly used, its principle is two pairs 5 of designs ' end adjacency, the primer that 3 ' extreme direction is opposite is in order to import change point.
Beneficial effect of the present invention is: adopt site-directed mutagenesis technique to transform the TEV proteinase gene, five amino acid residue in the specific change TEV proteolytic enzyme, to obtain the mutant of the higher TEV proteolytic enzyme of solubility and enzymic activity, strengthen the recombinant expressed application of its albumen in intestinal bacteria.
Description of drawings
Fig. 1 is TEV protease mutant TEV
Q(five mutant) and TEV proteolytic enzyme TEV
FThe Trimutant solubility relatively.
Fig. 2 is GSTcys fusion rotein and TEV proteolytic enzyme effect synoptic diagram.
Fig. 3 is TEV
FWith TEV
QThe activity ratio.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further detail.
Embodiment 1 utilizes rite-directed mutagenesis to obtain the TEV protease mutant
Design rite-directed mutagenesis primer is that template is carried out pcr amplification with the carrier (pET-TEV) that contains tobacco plaque virus protease gene, and reaction conditions is: 94 ℃ of 30s, and 55 ℃ of 30s, 72 ℃ of 6.5min circulate 30 times.The PCR product after DpnI digestion, 1% agarose gel electrophoresis.Cut glue purification, purified PCR product is carried out end smoothly reach 5 ' phosphoric acid (P) change processing, carry out self with efficient connection reagent Ligation Solution I again and connect transformed into escherichia coli DH5a competent cell, extraction mutant plasmid DNA.Simultaneously with five mutant called after TEV
Q, Trimutant called after TEV
F
Order-checking of embodiment 2 mutant genes and sequence alignment
To serve the sea by the mutant clone DH5a that aforesaid method obtains and give birth to the order-checking of worker's biotechnology Services Co., Ltd, sequencing result input BioEdit software, the relatively difference of gene base sequence and protein amino acid sequence before and after the sudden change.
The result shows that mutant protein and original TEV proteolytic enzyme are undergone mutation at five places totally.It is as follows that the DNA base of mutant and aminoacid sequence change situation:
Base is changed to: the 50th by the alternative C (cytosine(Cyt)) of G (guanine), the 166th by the alternative T (thymus pyrimidine) of G (guanine), the 202nd by the alternative A (VITAMIN B4) of G (guanine), the 229th by the alternative A (VITAMIN B4) of G (guanine), the 403rd substitutes T (thymus pyrimidine) by G (guanine), and the 404th substitutes C (cytosine(Cyt)) by G (guanine).
Correspondingly, amino acid is changed to: the 17th by Serine (S, Ser) replacement Threonine (T, Thr), the 56th by Xie Ansuan (V Val) replaces leucine (L, Leu), the 68th by aspartic acid (D Asp) replaces l-asparagine (N, Asn), the 77th by Xie Ansuan (V Val) replaces Isoleucine (I, Ile), the 135th by glycine (G, Gly) replace Serine (S, Ser).
Embodiment 3 TEV
QProteolytic enzyme and TEV
FProteolytic enzyme solubility and expression amount are relatively
With pET-TEV
QAnd pET-TEV
FRecombinant plasmid transformed intestinal bacteria Rosetta (DE3) competence.
1, a small amount of abduction delivering, relatively TEV
QProtease mutant and TEV
FThe variation of proteolytic enzyme solubility:
(1) picking list colony inoculation is in 5mL LB substratum (sulfur acid kantlex 25mg/mL, paraxin 25mg/mL), and the 220r/min shaking culture is spent the night in 37 ℃ of constant temperature shaking tables;
(2) add IPTG (final concentration is 1.0mmol/L), at 20 ℃ of constant temperature shaking table 220r/min abduction delivering 20h.
(3) manage the collection thalline with the 1.5mL doffer, wash and thalline is suspended in the 0.8mL lysis buffer again.
(4) resuspended liquid is centrifugal after ultrasonication, gets supernatant 120 μ L and adds 30 μ L5 * SDS-PAGE sample-loading buffer and prepare protein electrophoresis sample (supernatant).
(5) with the precipitation after the fragmentation after lysis buffer washing, be dissolved in 0.8mL 8mol/L urea soln again, get supernatant 120 μ L behind the recentrifuge and add 30 μ L, 5 * SDS-PAGE sample-loading buffer and prepare protein electrophoresis sample (precipitation).
(6) 12%SDS-PAGE electrophoretic analysis protein expression result.Xylene Brilliant Cyanine G R-250 dyeing.
SDS-PAGE electrophoresis detection result shows that solubility has obvious change, TEV before and after the TEV mutant proteinase
QThe solubility of protease mutant is improved.
2, a large amount of abduction deliverings and purifying, relatively TEV
QProtease mutant and TEV
FThe variation of proteolytic enzyme expression amount:
(1) picking list colony inoculation is in 5mL LB substratum, and the 220r/min shaking culture is spent the night in 37 ℃ of constant temperature shaking tables.
(2) be transferred in the 1L LB substratum with 1: 100 and cultivate OD
600About=0.6.
(3) add IPTG (final concentration is 1.0mmol/L), at 20 ℃ of constant temperature shaking table abduction delivering 20h.
(4) collect thalline, be suspended in again among the 30mL Lysis Buffer, resuspended liquid is centrifugal after ultrasonication, the Ni-NTA column purification.
(5) earlier with Lysis Buffer balance Ni-NTA chromatography column, will go up the cytoclasis supernatant liquor that the step obtains then and add in the Ni-NTA chromatography column, the control flow velocity is at 1mL/min.
(6) etc. after supernatant liquor has flowed,, use Washing buffer flush away foreign protein again with Lysis Buffer flushing.
(7) use Elution Buffer wash-out target protein at last, collect the about 30mL of target protein.
(8) after Elution Buffer wash-out finishes,, standby with Lysis Buffer balance Ni-NTA post.
(9) measure TEV respectively with the Bradford method
QProteolytic enzyme and TEV
FThe protein concentration of proteolytic enzyme calculates expression amount separately.
A large amount of abduction deliverings are measured TEV also with behind the Ni-NTA column purification respectively with the Bradford method
QProteolytic enzyme and TEV
FThe protein concentration of proteolytic enzyme calculates expression amount separately.Calculation result shows that the proteolytic enzyme expression amount is respectively 165mg (TEV in every liter of bacterium liquid
QProteolytic enzyme) and 108mg (TEV
FProteolytic enzyme).TEV
QThe expression amount of proteolytic enzyme is apparently higher than TEV
FProteolytic enzyme.
Embodiment 4 TEV
QProtease mutant and TEV
FThe proteolytic enzyme activity ratio
The enzymic activity of TEV proteolytic enzyme is measured by the substrate that its cutting contains the TEV protease recognition sequence, and we are substrate with fusion rotein GST-Cys.Endonuclease reaction carries out at 30 ℃ of thermostat water baths, and TEV proteolytic enzyme and GST-Cys fusion rotein ratio are 1: 10.
1. and 2. (1) draw isopyknic fusion rotein GST-Cys solution (200 μ L) in two 2mL doffer pipes, mark respectively.
(2) add the TEV proteolytic enzyme of equivalent respectively: TEV
FProteolytic enzyme (16 μ L), TEV
QProteolytic enzyme (12 μ L).
(3) replenish 50mM Tris-HCl damping fluid (50mM Tris-HCl pH 8.0,0.5mMEDTA, 1mM DTT) to 1600 μ L.
(4) in 30 ℃ of thermostat water baths, carry out endonuclease reaction
(5) take out 120 μ L respectively every 20min and prepare the protein electrophoresis sample.
(6) sample on the equivalent, the 12%SDS-PAGE enzyme analysis is cut the result.
Electrophoresis result shows TEV
QAlmost that the cutting of GST-Cys fusion rotein is complete behind the protease mutant 2h, and TEV
FProteolytic enzyme will reach same effect needs 3h.So TEV
QProtease mutant not only solubility is improved, and enzymic activity has also had very big improvement.
Sequence table
＜110〉Agricultural University Of Anhui
＜120〉TEV protease mutant and encoding gene and application thereof
<160>2
<170>PatentIn?version?3.3
<210>1
<211>708
<212>DNA
＜213〉artificial sequence
<220>
<221>gene
<222>(1)..(708)
<400>1
ggagaaagct?tgtttaaggg?gccgcgtgat?tacaacccga?tatcgagcag?catttgtcat 60
ttgacgaatg?aatctgatgg?gcacacaaca?tcgttgtatg?gtattggatt?tggtcccttc 120
atcattacaa?acaagcactt?gtttagaaga?aataatggaa?cactggtggt?ccaatcacta 180
catggtgtat?tcaaggtcaa?ggacaccacg?actttgcaac?aacacctcgt?tgatgggagg 240
gacatgataa?ttattcgcat?gcctaaggat?ttcccaccat?ttcctcaaaa?gctgaaattt 300
agagagccac?aaagggaaga?gcgcatatgt?cttgtgacaa?ccaacttcca?aactaagagc 360
atgtctagca?tggtgtcaga?cactagttgc?acattccctt?caggtgatgg?catattctgg 420
aagcattgga?ttcaaaccaa?ggatgggcag?tgtggcagtc?cattagtatc?aactagagat 480
gggttcattg?ttggtataca?ctcagcatcg?aatttcacca?acacaaacaa?ttatttcaca 540
agcgtgccga?aaaacttcat?ggaattgttg?acaaatcagg?aggcgcagca?gtgggttagt 600
ggttggcgat?taaatgctga?ctcagtattg?tgggggggcc?ataaagtttt?catggtgaaa 660
cctgaagagc?cttttcagcc?agttaaggaa?gcgactcaac?tcatgaat 708
<210>2
<211>236
<212>PRT
＜213〉artificial sequence
<220>
<221>DOMAIN
<222>(1)..(236)
<400>2
Gly?Glu?Ser?Leu?Phe?Lys?Gly?Pro?Arg?Asp?Tyr?Asn?Pro?Ile?Ser?Ser
1 5 10 15
Ser?Ile?Cys?His?Leu?Thr?Asn?Glu?Ser?Asp?Gly?His?Thr?Thr?Ser?Leu
20 25 30
Tyr?Gly?Ile?Gly?Phe?Gly?Pro?Phe?Ile?Ile?Thr?Asn?Lys?His?Leu?Phe
35 40 45
Arg?Arg?Asn?Asn?Gly?Thr?Leu?Val?Val?Gln?Ser?Leu?His?Gly?Val?Phe
50 55 60
Lys?Val?Lys?Asp?Thr?Thr?Thr?Leu?Gln?Gln?His?Leu?Val?Asp?Gly?Arg
65 70 75 80
Asp?Met?Ile?Ile?Ile?Arg?Met?Pro?Lys?Asp?Phe?Pro?Pro?Phe?Pro?Gln
85 90 95
Lys?Leu?Lys?Phe?Arg?Glu?Pro?Gln?Arg?Glu?Glu?Arg?Ile?Cys?Leu?Val
100 105 110
Thr?Thr?Asn?Phe?Gln?Thr?Lys?Ser?Met?Ser?Ser?Met?Val?Ser?Asp?Thr
115 120 125
Ser?Cys?Thr?Phe?Pro?Ser?Gly?Asp?Gly?Ile?Phe?Trp?Lys?His?Trp?Ile
130 135 140
Gln?Thr?Lys?Asp?Gly?Gln?Cys?Gly?Ser?Pro?Leu?Val?Ser?Thr?Arg?Asp
145 150 155 160
Gly?Phe?Ile?Val?Gly?Ile?His?Ser?Ala?Ser?Asn?Phe?Thr?Asn?Thr?Asn
165 170 175
Asn?Tyr?Phe?Thr?Ser?Val?Pro?Lys?Asn?Phe?Met?Glu?Leu?Leu?Thr?Asn
180 185 190
Gln?Glu?Ala?Gln?Gln?Trp?Val?Ser?Gly?Trp?Arg?Leu?Asn?Ala?Asp?Ser
195 200 205
Val?Leu?Trp?Gly?Gly?His?Lys?Val?Phe?Met?Val?Lys?Pro?Glu?Glu?Pro
210 215 220
Phe?Gln?Pro?Val?Lys?Glu?Ala?Thr?Gln?Leu?Met?Asn
225 230 235
Claims (6)
1.TEV protease mutant, it is characterized in that comparing with original TEV proteolytic enzyme, the 17th by Serine (Ser) replacement Threonine (Thr), the 56th by Xie Ansuan (Val) replacement leucine (Leu), the 68th by aspartic acid (Asp) replacement l-asparagine (Asn), the 77th replaces Isoleucine (Ile) by Xie Ansuan (Val), and the 135th replaces Serine (Ser) by glycine (Gly).
2. TEV protease mutant according to claim 1 is characterized in that described mutant has the aminoacid sequence shown in the sequence 2 in the sequence table.
3. the encode gene of TEV protease mutant as claimed in claim 1, its nucleotide sequence is shown in sequence in the sequence table 1.
4. one kind contains just like the described expression carrier of claim 3, it is characterized in that described expression vector is pET28b.
5. a host cell that contains described gene of claim 3 and the described carrier of claim 4 is characterized in that described host cell is intestinal bacteria Rosetta (DE3) competence.
6. the described TEV protease mutant of claim 1 carries out the recombinant expressed application of albumen in intestinal bacteria.
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Cited By (7)
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| CN103275917A (en) * | 2013-06-04 | 2013-09-04 | 南京师范大学 | TEV protease expression engineering bacteria and its construction and application |
| CN109055339A (en) * | 2018-09-19 | 2018-12-21 | 生工生物工程（上海）股份有限公司 | TEV protease mutant, gene, biomaterial, preparation method, reagent or kit and application |
| CN111019925A (en) * | 2018-10-10 | 2020-04-17 | 上饶市康可得生物科技有限公司 | Method for screening protease variants and protease variants obtained |
| CN111793638A (en) * | 2019-12-09 | 2020-10-20 | 贵州理工学院 | A recombinant TEV protease expression vector, expression system and application |
| CN113801866A (en) * | 2021-09-02 | 2021-12-17 | 无锡佰翱得生物科学有限公司 | High-efficiency expression recombinant TEV enzyme with high activity and stability, and preparation method, determination method and application thereof |
| JP2022504842A (en) * | 2018-10-10 | 2022-01-13 | 上饒市康可得生物科技有限公司 | TEV protease variants, fusion proteins thereof, preparation methods and uses |
| CN119799681A (en) * | 2024-12-31 | 2025-04-11 | 浙江玉安康瑞生物科技有限公司 | A recombinant TEV protease, a recombinant vector and an expression and purification method |
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| CN103275917A (en) * | 2013-06-04 | 2013-09-04 | 南京师范大学 | TEV protease expression engineering bacteria and its construction and application |
| CN109055339A (en) * | 2018-09-19 | 2018-12-21 | 生工生物工程（上海）股份有限公司 | TEV protease mutant, gene, biomaterial, preparation method, reagent or kit and application |
| CN109055339B (en) * | 2018-09-19 | 2020-09-01 | 生工生物工程（上海）股份有限公司 | TEV protease mutant, gene, biological material, preparation method, reagent or kit and application |
| CN111019925A (en) * | 2018-10-10 | 2020-04-17 | 上饶市康可得生物科技有限公司 | Method for screening protease variants and protease variants obtained |
| JP2022504842A (en) * | 2018-10-10 | 2022-01-13 | 上饒市康可得生物科技有限公司 | TEV protease variants, fusion proteins thereof, preparation methods and uses |
| CN111019925B (en) * | 2018-10-10 | 2022-08-23 | 上饶市康可得生物科技有限公司 | Method for screening protease variants and protease variants obtained |
| JP7296660B2 (en) | 2018-10-10 | 2023-06-23 | 上饒市康可得生物科技有限公司 | TEV protease variants, fusion proteins thereof, methods of preparation and uses |
| US11919936B2 (en) | 2018-10-10 | 2024-03-05 | Shangrao Concord Pharmaceutical Co., Ltd. | TEV protease variant, fusion protein thereof, preparation method therefor and use thereof |
| CN111793638A (en) * | 2019-12-09 | 2020-10-20 | 贵州理工学院 | A recombinant TEV protease expression vector, expression system and application |
| CN113801866A (en) * | 2021-09-02 | 2021-12-17 | 无锡佰翱得生物科学有限公司 | High-efficiency expression recombinant TEV enzyme with high activity and stability, and preparation method, determination method and application thereof |
| CN119799681A (en) * | 2024-12-31 | 2025-04-11 | 浙江玉安康瑞生物科技有限公司 | A recombinant TEV protease, a recombinant vector and an expression and purification method |
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