CN109136209A - Enterpeptidase light chain mutant and its application - Google Patents
Enterpeptidase light chain mutant and its application Download PDFInfo
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- CN109136209A CN109136209A CN201810788938.2A CN201810788938A CN109136209A CN 109136209 A CN109136209 A CN 109136209A CN 201810788938 A CN201810788938 A CN 201810788938A CN 109136209 A CN109136209 A CN 109136209A
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Abstract
The present invention relates to a kind of Enterpeptidase light chain mutant and its applications.Present invention discloses a kind of Enterpeptidase light chain mutant by sequence alterations, and compared with wild type, which has stronger enzymatic activity, and expression efficiency (including renaturation yield and rate of recovery) and stability greatly improve.
Description
Technical field
The invention belongs to bioengineering field, more particularly it relates to a kind of Enterpeptidase light chain mutant and its answer
With.
Background technique
Enterokinase (Enterokinase, EK) belongs to the protease of serine class, mainly appears on the 12 of mammal
In duodenum 12.The light chain of enterokinase and heavy chain Liang Ge subunit constitute its primary structure, catalytic activity of the light chain as enzyme
Center possesses the catalytic activity of holoenzyme, and can identify the amino acid sequence (Asp) for then digesting specificity4The site-Lys
(DDDDK), the peptide bond for then hydrolyzing carboxyl terminal after lysine cuts trypsinogen, thus activate trypsinogen,
Keep trypsase active.In its technology for being also widely used in fusion protein.
Natural enterokinase is the heterodimer connected by disulfide bond, the weight including a 82-140KDa size
The light chain of chain and a 35-62KDa size, two chains of enterokinase all include the carbohydrate of 30%-50%.Heavy chain can incite somebody to action
Enterokinase is anchored on goldbeater's skin.Also comprising the transmembrane domain of an amino terminal in heavy chain, in terms of identifying small-molecular peptides
Influence very little, but to identification specific small molecule substance and inhibitor in terms of have a significant impact.Light chain is then enterokinase
Catalytic active center, the serine protease domain comprising a chymotrypsin sample, amino acid (Asp)4- Lys sequence it is special
The identification of one property and enzymatic hydrolysis are all realized by light chain.
During producing recombinant protein using technique for gene engineering, in order to improve destination protein production and purifying
Efficiency often uses this method of amalgamation and expression.Amalgamation and expression, which refers to, is connected target gene with foreign gene, produces after expression
The stability of object increases, and the fusion segment in amalgamation and expression rear fusion protein is often with different labels, significantly
The easy purifying of albumen after manufacturing.But due to increased on destination protein fusion segment after, the life of destination protein
Object function is often affected, so destination protein can not be used in the form of fusion protein, fusion segment must be gone
Except the biological function of destination protein competence exertion itself later.The method of fusion segment is in the removal fusion protein used earliest
Chemical cleavage method, but because the specificity of cracking site is low, cracking may occur in other sites, to make destination protein
Generate unnecessary modification.Increasingly developed with zymetology, chemical cleavage method is gradually replaced enzymatic isolation method.Use enzymatic isolation method
Removal fusion segment needs to design the recognition site for having specificity, protease by identification be located at destination protein and label protein it
Between specific position after carry out specific cutting, the destination protein obtained after cutting has bioactivity.Fibrin ferment, blood coagulation
Factor Xa and enterokinase are the protease for being most often used to cleavage of fusion proteins, wherein enterokinase because its specific highest
What is used is the most extensive.
Enterokinase as a kind of ideal toolenzyme, can by the sequence of identification specificity come cleavage of fusion proteins to
Achieve the purpose that produce destination protein, and it is residual in the amino terminal of destination protein to will not leave behind any unnecessary amino acid
Base.Certainly, the nickase as a kind of locus specificity, the effect that enterokinase is played in cleavage of fusion proteins will receive accidentally
The obstruction in the cutting in other sites occurs, these sites are made of acidic residues and alkaline residue.
The light chain of enterokinase as catalytic subunit not only possesses whole activity of holoenzyme, but also can it is several not
With cell line in express, including Chinese hamster ovary celI, Pichia pastoris, saccharomyces cerevisiae, black-koji mould and Escherichia coli etc. all can be with
Successfully expression Enterpeptidase light chain.Since the reaction condition of the enzymatic hydrolysis of enterokinase is relatively mild, so cause to be cut
Protein is very low in the probability that other non-cutting positions are broken.Furthermore ten dtex of site of enterokinase cleavage of fusion proteins
Very, be after whole identification sequences, will not demolition purpose albumen first place amino acid sequence, keep the first amino acid complete
Meet native protein entirely.
All features of comprehensive enterokinase, more and more genetic engineering pharmaceuticals can select enterokinase as in downstream purification
The toolenzyme when albumen of amalgamation and expression.However, natural enterokinase has unstable characteristic, it is not easy to purify and saves,
So that its application is subject to certain restrictions.Meanwhile the production efficiency and enzymatic activity of recombinant production enterokinase are also urgently further
Raising.
Summary of the invention
The purpose of the present invention is to provide a kind of Enterpeptidase light chain mutant and its applications.
In the first aspect of the present invention, a kind of Enterpeptidase light chain mutant is provided, amino acid sequence is corresponding to SEQ
62nd amino acids of ID NO:1 amino acid sequence sport threonine, and the 177th amino acids sport glutamic acid, Yi Ji
112 amino acids sport threonine or alanine.
In a preferred embodiment, the 112nd amino acids sport threonine.
In another preferred example, the Enterpeptidase light chain mutant further include: the amino acid as shown in SEQ ID NO:1
Sequence is by replacing, missing or adding one or several (such as 1-10, preferably 1-5, more preferably 1-3) amino acid and tool
There are the derivative albumen of the identical function of Enterpeptidase light chain mutant of aforementioned definition, and the 62nd bit amino of its amino acid sequence
Acid is threonine, and the 177th amino acids are glutamic acid and the 112nd amino acids are threonine or alanine.
In another preferred example, the Enterpeptidase light chain mutant further include: with amino acid shown in SEQ ID NO:1
Sequence is with 85% or more (as having 90% or more, 95% or more, 98% or more or 99% or more) Amino acid sequence identity
And the identical function of Enterpeptidase light chain mutant with aforementioned definition derivative albumen, and the of its amino acid sequence
62 amino acids are threonine, and the 177th amino acids are glutamic acid and the 112nd amino acids are threonine or alanine.
In another aspect of this invention, a kind of isolated polynucleotides are provided, which is selected from the group:
(i) polynucleotides of the coding Enterpeptidase light chain mutant；Or
(ii) polynucleotides complementary with the polynucleotides in (i).
In another aspect of this invention, a kind of carrier is provided, it contains the polynucleotides.
In another aspect of this invention, a kind of genetically engineered host cell is provided, it contains the carrier or base
Because of the polynucleotides described in being integrated in group.
In another aspect of this invention, a kind of method producing the Enterpeptidase light chain mutant is provided, comprising steps of
(1) the culture host cell obtains culture；With
(2) the Enterpeptidase light chain mutant is separated from culture.
In another aspect of this invention, a kind of side for improving Enterpeptidase light chain production efficiency, enzymatic activity or stability is provided
Method, which comprises for the Enterpeptidase light chain of wild type, will correspond to the 62nd of SEQ ID NO:1 amino acid sequence
Amino acid mutation is threonine, the 177th amino acids sport glutamic acid and the 112nd amino acids sport threonine or
Alanine.
In another aspect of this invention, a kind of composition is provided, it includes the Enterpeptidase light chain mutant, Yi Jiyu
The compatible carrier of the Enterpeptidase light chain mutant.
In another aspect of this invention, the purposes of the Enterpeptidase light chain mutant or the composition is provided, is used
In cutting peptide chain；Preferably, for identification (Asp) in peptide chain4The peptide bond of carboxyl terminal after the site-Lys, hydrolysis lysine.
In another aspect of this invention, provide it is a kind of for cutting the kit of peptide chain, including the enterokinase
Light chain mutant.
In another aspect of this invention, provide it is a kind of for cutting the kit of peptide chain, including the combination
Object.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Detailed description of the invention
Fig. 1, EK surface discontinuity Locus Analysis in Shoots.
The surface site and charge schematic diagram of the A62 and A177 of A and B, wild type EK；
The surface site and charge schematic diagram of C and D, mutant A62T-A177E.
Fig. 2, bacterium colony PCR qualification result；Each swimming lane applied sample amount is 10 μ L.
M, DNA molecular amount standard (bp)；
Swimming lane 1, BL21 (DE3) transformed bacteria 1；
Swimming lane 2, BL21 (DE3) transformed bacteria 2；
Swimming lane 3, BL21 (DE3) transformed bacteria 3；
Swimming lane 4, BL21 (DE3) transformed bacteria 4；
Swimming lane 5, BL21 (DE3) transformed bacteria 5.
The inducing expression of Fig. 3, EK mutant strain；Each swimming lane applied sample amount is 10 μ L.
M, albumen marker (kDa)；
Swimming lane 1, before BL21 (DE3) inducing expression；
Swimming lane 2, EK mutant strain is after inducing expression；
Swimming lane 3, supernatant after EK mutant strain ultrasonication；
Swimming lane 4 precipitates after EK mutant strain ultrasonication.
Fig. 4, DEAE column purification albumen；Each swimming lane applied sample amount is 10 μ L.
M, Protein Marker (kDa)；
Swimming lane 1, before loading；
Swimming lane 2, loading is pierced by liquid；
Swimming lane 3, balance are pierced by liquid；
The 4~14, the 1st~11 pipe eluent of swimming lane.
Specific embodiment
The present inventor is dedicated to improving the stability and activity of enterokinase, by long-term research and screens, obtains one
Kind passes through the Enterpeptidase light chain mutant of sequence alterations, and compared with wild type, which has stronger enzymatic activity, expression effect
Rate (including renaturation yield and rate of recovery) and stability greatly improve.The present invention is completed on this basis.
As used herein, unless otherwise stated, " the Enterpeptidase light chain mutant ", " mutain EK light chain ", " prominent
Variant EK light chain " etc. is used interchangeably, and each means that amino acid sequence corresponds to the 62nd ammonia of SEQ ID NO:1 amino acid sequence
Base acid mutation is threonine, the 177th amino acids sport glutamic acid and the 112nd amino acids sport threonine or third
The albumen of propylhomoserin.
If desired indicate wild type albumen, will be denoted as " wild type Enterpeptidase light chain ", " Enterpeptidase light chain " or
" wild-type protein ", amino acid sequence are SEQ ID NO:1.
As used herein, described " Enterpeptidase light chain (or mutant) activity " is defined with the unit of activity of enzyme.
One unit of activity (1U) of enzyme is defined as 25 DEG C, pH7.4) under the conditions of, reaction system 3.0ml (1cm optical path) is digested per minute
N- Benzoyl-L-arginine ethyl ester (N-benzoyl-L-arginine ethyl ester, BAEE) makes the absorption value under 253nm
Increase by 0.001 and is defined as a BAEE unit.
As used herein, " separation " it is (former if it is crude to refer to that substance is separated from its primal environment
Beginning environment is natural surroundings).As under the native state in active somatic cell polynucleotide and albumen be not isolate and purify
, but same polynucleotide or albumen such as from native state with separated in other existing substances, then to isolate and purify
's.
As used herein, " isolated Enterpeptidase light chain mutant " refers to Enterpeptidase light chain mutant substantially free of natural
Relative other albumen, lipid, carbohydrate or other materials.Those skilled in the art can use the protein purification skill of standard
Art purifies Enterpeptidase light chain mutant.Substantially pure albumen can generate single master in non-reducing polyacrylamide gel
Band.
As used herein, " recombination " refers to by genetic engineering means the albumen for obtaining (or a large amount of preparations), gene
Engineered vector or cell etc..
Albumen of the invention can be recombinant protein, native protein, synthetic proteins, preferably recombinant protein.Egg of the invention
It is white to can be native purified product or chemically synthesized product, or use recombinant technique from protokaryon or eucaryon host (example
Such as, bacterium, yeast, higher plant, insect and mammalian cell) in generate.According to host used in recombinant production scheme, originally
The albumen of invention can be glycosylated, or can be nonglycosylated.Albumen of the invention may also include or not include starting
Methionine residues.
The invention also includes segment, the derivative and analogue of the Enterpeptidase light chain mutant.As used herein, term
" segment ", " derivative " and " analog ", which refers to, is kept substantially the identical biology of natural enterokinase light chain mutant of the invention
Learn function or active albumen.Protein fragments of the invention, derivative or the like can be (i) have it is one or more conservative or
Non-conservative amino acid residue (preferably conservative amino acid) substituted albumen, and such substituted amino acid residue
Can be may not be by genetic code encoding, or (ii) has substituent group in one or more amino acid residues
Albumen, or (iii) maturation protein and another compound (for example extending the compound of protein half-life, such as polyethylene glycol) melt
Conjunction is formed by albumen, or (iv) additional amino acid sequence is fused to this protein sequence and albumen (such as leader sequence that is formed
Secretion sequence or for purifying the sequence of this albumen or proprotein sequence or fusion protein).According to these pieces of the definition of this paper
Section, derivative and analogue belong to scope known to those skilled in the art.However, the Enterpeptidase light chain mutant
The 62nd bit amino and its in the amino acid sequence of segment, derivative and analogue, corresponding to SEQ ID NO:1 amino acid sequence
Acid is threonine, and the 177th amino acids are glutamic acid and the 112nd amino acids are threonine or alanine.
In the present invention, term " Enterpeptidase light chain mutant " further includes with light with enterokinase verified in embodiment
Other sequence variations forms of chain mutant identical function.These variant forms include (but being not limited to): several are (usually
1-20, more preferably 1-10, also more preferably such as 1-8,1-5,1-3 or 1-2) missing of amino acid, insertion and/or
Replace, and C-terminal and/or N-terminal addition or lack it is one or several (usually within 20, preferably 10 with
It is interior, be more preferably within 5) amino acid.For example, in the art, when being substituted with similar nature or similar amino acid,
The function of protein is not usually changed.For another example, in C-terminal and/or N-terminal addition or really, one or several amino acid are logical
The function of protein will not often be changed.The term further includes the active fragment and reactive derivative of Enterpeptidase light chain mutant.
Best, in these variant forms, the 62nd amino acids corresponding to SEQ ID NO:1 amino acid sequence are threonine, the
177 amino acids are glutamic acid and the 112nd amino acids are threonine or alanine.
Invention also provides the analog of Enterpeptidase light chain mutant or albumen.These analogs and natural Enterpeptidase light chain are prominent
The difference of variant can be the difference on amino acid sequence, be also possible to not influence the difference on the modified forms of sequence, or
Have both at the same time.These albumen include natural or induction genetic variant.Induction variant can be obtained by various technologies, such as
Random mutagenesis is generated and radiating or being exposed to mutagens, can also pass through site-directed mutagenesis or other known molecular biology
Technology.Analog further includes the analog with the residue (such as D- amino acid) different from natural L-amino acids, and is had non-
The analog of natural or synthetic amino acid (such as β, gamma-amino acid).It should be understood that albumen of the invention is not limited to
State the representative albumen enumerated.
Modification (not changing primary structure usually) form includes: the chemical derivative form such as acetyl of internal or external albumen
Change or carboxylated.Modification further includes glycosylation.Modified forms further include with phosphorylated amino acid residue (such as phosphotyrosine,
Phosphoserine, phosphothreonine) sequence.It further include being modified to improve its anti-proteolytic properties or optimize molten
Solve the albumen of performance.
The present invention also provides the polynucleotides for encoding Enterpeptidase light chain mutant of the present invention or its conservative variation's albumen
Sequence.
Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people
The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.
The polynucleotides for encoding mutant maturation protein of the invention include: a coded sequence for encoding mature albumen；At
The white coded sequence of soft-boiled eggs and various additional coding sequences；The coded sequence (and optional additional coding sequence) of maturation protein with
And non-coding sequence.
The term polynucleotides of albumen " coding " can be the polynucleotides including encoding this albumen, be also possible to further include
The polynucleotides of additional code and/or non-coding sequence.
The invention further relates to the variant of above-mentioned polynucleotides, coding has the egg of identical amino acid sequence with the present invention
White or albumen segment, analogs and derivatives.The variant of this polynucleotides can be the allelic variant naturally occurred or
The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insertion variant.Such as this
Known to field, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution,
Missing or insertion, but not from substantially change its encode albumen function.
Enterpeptidase light chain mutant nucleotide full length sequence or its segment of the invention can usually use PCR amplification method, again
Group method or artificial synthesized method obtain.For PCR amplification method, can disclosed related nucleotide sequence according to the present invention, especially
It is that open reading frame sequence carrys out design primer, and with the commercially available library cDNA or presses conventional method well known by persons skilled in the art
The prepared library cDNA expands as template and obtains related sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly
Then the segment that each time amplifies is stitched together by PCR amplification by proper order again.
Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical
After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.
At present, it is already possible to obtain encoding albumen of the present invention (or its segment or its derivative by chemical synthesis completely
Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and
In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.
The present invention also relates to the carriers comprising polynucleotides of the invention, and with carrier or Enterpeptidase light chain of the invention
The genetically engineered host cell of mutant code sequence, and generate through recombinant technique the side of albumen of the present invention
Method.
By the recombinant dna technology of routine (Science, 1984；224:1431), using polynucleotide of the invention
Sequence come express or produce recombination Enterpeptidase light chain mutant.In general there are following steps:
(1) polynucleotides (or variant) of coding Enterpeptidase light chain mutant of the invention, or with containing the multicore
The recombinant expression carrier of thuja acid converts or suitable host cell of transduceing；
(2) host cell that is cultivated in suitable culture medium；
(3) be separated from culture medium or cell, protein purification.
In the present invention, Enterpeptidase light chain mutant polynucleotide sequence be can be plugged into recombinant expression carrier.Term " recombination
Expression vector " refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus
Or other carriers.As long as any plasmid and carrier can be used in short, can replicate and stablize in host.Expression vector
One important feature is to usually contain replication orgin, promoter, marker gene and translation control element.
Method well-known to those having ordinary skill in the art can be used to construct the DNA sequence dna of mutant code containing Enterpeptidase light chain and conjunction
Suitable transcription/translation control signal expression vector.These methods include recombinant DNA technology in vi, DNA synthetic technology, in vivo
Recombinant technique etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.
Expression vector further includes the ribosome bind site and transcription terminator of translation initiation.
In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion
The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg
White (GFP), or kanamycins or amicillin resistance for Escherichia coli.
Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for converting suitable
When host cell, allow it to expression protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high
Equal eukaryocytes, such as plant cell.Representative example has: Escherichia coli, streptomyces, Agrobacterium；Fungal cell's such as yeast；It plants
Object cell etc..
When polynucleotides of the invention are expressed in higher eucaryotic cells, if will when being inserted into enhancer sequence in the carrier
Transcription can be made to be enhanced.Enhancer is the cis-acting factors of DNA, generally about has 10 to 300 base-pairs, acts on and open
Mover is to enhance the transcription of gene.
Persons skilled in the art are aware that how to select carrier, promoter, enhancer and host cell appropriate.
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original
When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is using MgCl2.If desired, conversion can also use the side of electroporation
Method carries out.When host is eucaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, express the albumen of coded by said gene of the invention.According to used
Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth
It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction)
Cell is further cultured for a period of time by the promoter for inducing selection.
As another example of the invention, Enterpeptidase light chain mutant is produced by genetic engineering means, for example utilize
Any suitable genetic engineering bacterium production Enterpeptidase light chain mutant, separates the Enterpeptidase light chain mutant.
Recombinant protein in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as
Fruit needs, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This
A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process is used
Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales
The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.
In a specific embodiment of the present invention, the present invention has recombinantly expressed the light chain mutant of enterokinase, prominent by screening
Point is conjugated, active height, the high ox intestine kinase mutant of stability are obtained.The heavy chain and light chain of enterokinase pass through a pair of of disulfide bond
Connection.Firstly, the light chain of the present inventor's single expression enterokinase, by 112 connect with heavy chain cysteine mutation therein
It for threonine or alanine, recombinantly expresses, after purification, obtains the mutant in the serial site, improve renaturation yield, increase steady
Qualitative, it is optimal that the screening discovery of different aminoacids sports threonine.On this basis, the present inventor increases mutational site
The bis- mutation of A62T and A177E, obtain Trimutant EK-C112T-A62T-A186E, the EK-C112A-A62T-A177E of EK, make
The stability for obtaining EK further increases.
In a specific embodiment of the present invention, find that enterokinase is prominent by the activity and other properties of measurement mutation enterokinase
For variant compared with natural enterokinase, activity and stability have extremely significant raising.
Enterpeptidase light chain mutant of the invention has good renaturation yield, the polypeptide of recombinant expression after recombinant expression
With good stability and enzymatic activity.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or
According to the normal condition proposed by manufacturer.
Embodiment 1, ox intestine kinase mutant
The amino acid sequence of the light chain of wild type ox intestine kinase (EK) is following (SEQ ID NO:1):
It is as a result sent out for each amino acid sites of EK light chain present inventor has performed largely analyzing, comparing and testing
Now positioned at two hydrophobic amino acid Ala-62 and Ala-177 on protein three-dimensional structure surface, such as Fig. 1, the R base of the amino acid
It is not formed and is interacted with other amino acid side chains in protein structure for a hydrogen atom.
The amino acid of Ala-62 and the two sites Ala-177 are sported threonine and glutamic acid by the present inventor respectively,
The hydrophily and partial charge that EK protein surface can be increased can increase the stability of EK in the solution, and it is multiple can also to improve it
Property rate.
Meanwhile the quasi- mutation for carrying out Cys-112 of the present inventor, to improve purification and recovery rate, renaturation yield or stability.
In subsequent embodiment, the effect of each mutant is verified.
The PCR identification of embodiment 2, colonies
Synthesize wild type gene and its mutant gene, wild type ammonia of the mutant gene coding in embodiment 1
On the basis of base acid sequence, the mutant polypeptide of following site mutation: C112A, C112T, C112A-A62T-A177E occurs,
C112T-A62T-A177E。
The mutant gene of synthesis is constructed in the multiple cloning sites of expression plasmid pET28a, the recombinant plasmid of synthesis turns
Change expression strain BL21 (DE3), coated plate, 37 DEG C of overnight incubations, the preferable single bacterium of growing way falls on 3mL containing blocking that in picking reformer plate
In the LB culture medium of resistance, 37 DEG C shaking table culture 5~6 hours, carry out bacterium colony PCR when the muddy naked eyes of bacterium solution are visible.According to bacterium
PCR system is fallen successively to be loaded.
PCR system: totally 20 μ L
PCR condition
Bacterium colony PCR qualification result such as Fig. 2 of bovine enterokinase light chain mutant is converted (with C112T-A62T-A177E mutant
For illustrate).The band of two positions is clear that from figure, a band clearly becomes clear, another band then phase
To relatively fuzzy.Recombinant plasmid is transferred in BL21 when conversion, and Fig. 2 is the qualification result for being transferred to BL21.Clearly bright stripe size
For 800bp or so, it is consistent with the theoretical value of required recon, the size of the band of another location is 100bp or so, be should be
Primer dimer band.The single colonie of all pickings is by being recon after bacterium colony PCR identification.
Embodiment 3, inducing expression
Bovine enterokinase light chain mutant C112T-A62T-A177E is subjected to inducing expression, is induced with the IPTG of 1mM.
Detected by SDS-PAGE protein electrophoresis, after induction band compared with inducing preceding article band at 30kDa size slightly
It thickens, supernatant band obviously thickens at 30kDa size without significant change, precipitation band after ultrasonication, this is expressed as wrapping
Contain body surface to reach.Such as Fig. 3 (by taking C112T-A62T-A177E mutant as an example).
Embodiment 4, enterokinase renaturing inclusion bodies
Inclusion body is collected by centrifugation after bacterial cell disruption, the Tris- of the triton x-100 containing 0.5% is added according to 1g:10ml
Inclusion body is collected by centrifugation in HCl pH of buffer 8.0,50mM, sufficiently suspension 1h, and it is slow that the Tris-HCl without triton x-100 is added
The washing of fliud flushing removes remaining triton x-100, the inclusion body purified.Take inclusion body according to the every ml 8M of 10mg weight in wet base
Urea dissolves, and after 1h, DTT 5mM, settling flux 1h is added, and being slowly added into renaturation solution according to 1:10 volume ratio, (Tris-HCl is slow
Fliud flushing pH8.5,50mM, 1mM GSH, 1mM GSSG) in.After placing renaturation 20-30h, detection activity, purifying.
Embodiment 5, DEAE ion column purification
It is packed into DEAE glue in pillar, is first rinsed with deionized water, thoroughly washes away remaining ethyl alcohol, then use 0.1M
NaOH handles 5 column volumes, is rushed pillar to neutrality with water, rinses 2 column volumes with 1M NaCl, is then rushed with deionized water
3 column volumes are washed, finally balance 5-10 column volume with equilibrium liquid 50mM Tris-HCl PH8.5.By 10 minutes 1 column volumes
Loading.After end of the sample, 5 column volumes are balanced with 50mM Tris-HCl PH8.5 buffer.With 0~500mM after balance
NaCl linear elution collects eluent according to column volume.Such as Fig. 4 (by taking C112T-A62T-A177E mutant as an example).
The enzyme activity determination of embodiment 6, enterokinase
Using the activity of BAEE substrate method measurement bovine enterokinase light chain mutant.Prepare the potassium dihydrogen phosphate 13mL of 0.067M
The buffer formed with the disodium hydrogen phosphate 87mL of 0.067M, is settled to 50mL for appropriate BAEE substrate, takes 10mL to be added to slow
In fliud flushing, false bottom object will be surveyed and be previously placed in 25 DEG C of water-baths.It takes the substrate of 3mL in quartz colorimetric utensil, is added appropriate to be measured
Enzyme mixes, and the registration that spectrophotometer changes in 3 minutes is recorded at 253nm.OD variation 0.001 per minute is living for a BAEE
Property unit.
Embodiment 7, stability contrast
EK light chain mutant series protein purification result such as table 1.
Table 1
Through comparing, in the purification process of the EK light chain after renaturation, the purification and recovery rate of Trimutant is mentioned compared with single mutation
2 times high, specific activity also increases about 1.5 times.The renaturation yield and stability for illustrating Trimutant are extremely significantly improved, and are presented
The unexpected effect compared with before mutation out.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Shanghai Yaxin Biotech Co., Ltd.
<120>Enterpeptidase light chain mutant and its application
<130> 182480
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 235
<212> PRT
<213>ox (Bovine)
<400> 1
Ile Val Gly Gly Ser Asp Ser Arg Glu Gly Ala Trp Pro Trp Val Val
1 5 10 15
Ala Leu Tyr Phe Asp Asp Gln Gln Val Cys Gly Ala Ser Leu Val Ser
20 25 30
Arg Asp Trp Leu Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn Met
35 40 45
Glu Pro Ser Lys Trp Lys Ala Val Leu Gly Leu His Met Ala Ser Asn
50 55 60
Leu Thr Ser Pro Gln Ile Glu Thr Arg Leu Ile Asp Gln Ile Val Ile
65 70 75 80
Asn Pro His Tyr Asn Lys Arg Arg Lys Asn Asn Asp Ile Ala Met Met
85 90 95
His Leu Glu Met Lys Val Asn Tyr Thr Asp Tyr Ile Gln Pro Ile Cys
100 105 110
Leu Pro Glu Glu Asn Gln Val Phe Pro Pro Gly Arg Ile Cys Ser Ile
115 120 125
Ala Gly Trp Gly Ala Leu Ile Tyr Gln Gly Ser Thr Ala Asp Val Leu
130 135 140
Gln Glu Ala Asp Val Pro Leu Leu Ser Asn Glu Lys Cys Gln Gln Gln
145 150 155 160
Met Pro Glu Tyr Asn Ile Thr Glu Asn Met Val Cys Ala Gly Tyr Glu
165 170 175
Ala Gly Gly Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Met
180 185 190
Cys Gln Glu Asn Asn Arg Trp Leu Leu Ala Gly Val Thr Ser Phe Gly
195 200 205
Tyr Gln Cys Ala Leu Pro Asn Arg Pro Gly Val Tyr Ala Arg Val Pro
210 215 220
Arg Phe Thr Glu Trp Ile Gln Ser Phe Leu His
225 230 235
Claims (10)
1. a kind of Enterpeptidase light chain mutant, which is characterized in that its amino acid sequence is corresponding to SEQ ID NO:1 amino acid sequence
62nd amino acids of column sport threonine, and the 177th amino acids sport glutamic acid and the mutation of the 112nd amino acids
For threonine or alanine.
2. Enterpeptidase light chain mutant as described in claim 1, which is characterized in that the 112nd amino acids sport
Threonine.
3. a kind of isolated polynucleotides, which is characterized in that the polynucleotides are selected from the group:
(i) polynucleotides of Enterpeptidase light chain mutant described in claim 1 are encoded；Or
(ii) polynucleotides complementary with the polynucleotides in (i).
4. a kind of carrier, which is characterized in that it contains polynucleotides as claimed in claim 3.
5. a kind of genetically engineered host cell, which is characterized in that it contains carrier or genome as claimed in claim 4
In be integrated with polynucleotides as claimed in claim 3.
6. a kind of method for producing Enterpeptidase light chain mutant described in claim 1, which is characterized in that comprising steps of
(1) host cell described in claim 5 is cultivated, culture is obtained；With
(2) Enterpeptidase light chain mutant described in claim 1 is separated from culture.
7. a kind of method for improving Enterpeptidase light chain production efficiency, enzymatic activity or stability, which is characterized in that the method packet
Include: for the Enterpeptidase light chain of wild type, the 62nd amino acids that will correspond to SEQ ID NO:1 amino acid sequence sport Soviet Union
Propylhomoserin, the 177th amino acids sport glutamic acid and the 112nd amino acids sport threonine or alanine.
8. a kind of composition, it includes Enterpeptidase light chain mutant of any of claims 1 or 2, and light with the enterokinase
The compatible carrier of chain mutant.
9. the purposes of Enterpeptidase light chain mutant of any of claims 1 or 2 or composition according to any one of claims 8, for cutting
Cut peptide chain；Preferably, for identification (Asp) in peptide chain4The peptide bond of carboxyl terminal after the site-Lys, hydrolysis lysine.
10. a kind of for cutting the kit of peptide chain, which is characterized in that include: in the kit
Enterpeptidase light chain mutant of any of claims 1 or 2；Or
Composition according to any one of claims 8.
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Cited By (2)
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| CN114774397A (en) * | 2022-06-20 | 2022-07-22 | 北京惠之衡生物科技有限公司 | Bovine enterokinase light chain protein mutant and recombinant fusion protein |
| CN114807101A (en) * | 2022-06-20 | 2022-07-29 | 北京惠之衡生物科技有限公司 | Fusion protein containing bovine enterokinase light chain protein, expression vector and recombinant engineering bacteria thereof |
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| CN101386847A (en) * | 2007-05-11 | 2009-03-18 | 上海张江生物技术有限公司 | Recombinant bovine enterokinase, preparation method and use thereof |
| WO2013092855A1 (en) * | 2011-12-23 | 2013-06-27 | Novo Nordisk A/S | Modified enterokinase light chain |
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| WO2013092855A1 (en) * | 2011-12-23 | 2013-06-27 | Novo Nordisk A/S | Modified enterokinase light chain |
| CN104561063A (en) * | 2015-01-27 | 2015-04-29 | 河北科技大学 | cDNA fragment, high-efficiency expression recombinant plasmid and genetically engineered bacterium of bovine enterokinase and and expression method of genetically engineered bacterium |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114774397A (en) * | 2022-06-20 | 2022-07-22 | 北京惠之衡生物科技有限公司 | Bovine enterokinase light chain protein mutant and recombinant fusion protein |
| CN114807101A (en) * | 2022-06-20 | 2022-07-29 | 北京惠之衡生物科技有限公司 | Fusion protein containing bovine enterokinase light chain protein, expression vector and recombinant engineering bacteria thereof |
| CN114807101B (en) * | 2022-06-20 | 2022-09-16 | 北京惠之衡生物科技有限公司 | Fusion protein containing bovine enterokinase light chain protein, expression vector and recombinant engineering bacteria thereof |
| CN116064491A (en) * | 2022-06-20 | 2023-05-05 | 北京惠之衡生物科技有限公司 | Bovine enterokinase light chain protein mutant and recombinant fusion protein thereof |
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