CN106337043B - People's trypsin mutant of high stability - Google Patents
People's trypsin mutant of high stability Download PDFInfo
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- CN106337043B CN106337043B CN201510418411.7A CN201510418411A CN106337043B CN 106337043 B CN106337043 B CN 106337043B CN 201510418411 A CN201510418411 A CN 201510418411A CN 106337043 B CN106337043 B CN 106337043B
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Abstract
The present invention relates to people's trypsin mutants of high stability.The inventors discovered that some sites of people's anion tryptose are related to the stability of heat or pH value to it, the site is the 121st or the 122nd, a kind of people's anion tryptose mutant is obtained on this basis, compared with wild type, the mutant stability with higher.Also, the 139th and 206 double mutation that people's anion tryptose is combined on the basis of aforementioned mutation, so that the stability and enzymatic activity of enzyme are even more ideal.
Description
Technical field
The invention belongs to field of biotechnology；More particularly it relates to people's trypsin mutant of high stability.
Background technique
Since extracorporeal recombination is applied so far from the maturation seventies, a large amount of vivo protein is by recombinant clone.Recombinate egg
It is white compared to the albumen extracted out of animal body, have several big apparent advantages: not being related to animal origin first, non-animal derived property is raw
Production process；Secondly, background understands, purity is high is suitble to reconstituted drug production, free serum culture etc..
For trypsase in clinical immunotherapy of tumors, tissue cultures, the processes such as production of vaccine for man are indispensable,
Current trypsase be it is animal derived, have virus pollute a possibility that, so as to cause people store altogether illness propagation, using no animal
The recombinant trypsin of source property is to solve the problems, such as this basic scheme.
In 2014, United States Pharmacopeia gave recombination pancreas for the recombinant trypsin during being applied to bio-pharmaceuticals
The newest examination criteria of protease.In skin tissue cell incubation, blueness that the product of non-animal derived property is increasingly subject to
It looks at.Went et al. is thin with the cuticula in animal derived trypsase and recombinant trypsin the digestion epidermis extracted respectively
Born of the same parents.Show that the growing state of skin tissue cell after recombinant trypsin digestion will be substantially better than animal derived pancreatin treated
Cell.
To sum up, this field needs to be mass produced the trypsase in people source.But the work of natural people's trypsase enzyme
Property is low, stability is poor, greatly limits its popularization and application.Therefore, there is an urgent need in the art to find to improve the effective of its stability
Means.
Summary of the invention
The purpose of the present invention is to provide a kind of people's trypsin mutants of high stability.
In the first aspect of the present invention, the mutant of people's anionic trypsin, the amino acid sequence of the mutant are provided
Column correspond to SEQ ID NO:1, and the 121st sports Ala and/or the 139th by Ser and sport Cys by Ser.
In a preferred embodiment, the amino acid sequence of the mutant further include: correspond to SEQ ID NO:1, the 206th
Cys is sported by Ser；Or the 122nd sport Leu by Arg；Also, the mutant is not amino acid sequence such as SEQ
ID NO:1 amino acid sequence and the 139th sport Cys, the 206th mutant for sporting by Ser Cys by Ser.
In another preferred example, the mutant is:
Such as the amino acid sequence of SEQ ID NO:1, but wherein the 139th by Ser sport Cys, the 206th dashed forward by Ser
Become Cys, the 121st sport Ala the 122nd by Ser and Leu (abbreviation S139C-S206C-S121A- sported by Arg
R122L)；Or
Such as the amino acid sequence of SEQ ID NO:1, but wherein the 139th by Ser sport Cys, the 206th dashed forward by Ser
Become Cys and the 122nd and Leu (abbreviation S139C-S206C-R122L) is sported by Arg；Or
Such as the amino acid sequence of SEQ ID NO:1, but wherein the 139th by Ser sport Cys, the 206th dashed forward by Ser
Become Cys and the 121st and Ala (abbreviation S139C-S206C-S121A) is sported by Ser；Or
Such as the amino acid sequence of SEQ ID NO:1, but wherein the 121st sport Ala and the 122nd by Ser and dashed forward by Arg
Become Leu (abbreviation S121A-R122L).
In another preferred example, S139C-S206C-S121A-R122L is in pH3-11,25 DEG C of preservation 2h, and the vigor of enzyme is equal
It is maintained at 80% or more；PH3,60 DEG C of preservation 12h remnant enzyme activities may remain in 70% or more；Its high temperature resistance and pH tolerance
Property is splendid.
In another aspect of this invention, the polynucleotides of separation, mutant described in the polynucleotide encoding are provided.
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 of mutant producing people's anionic trypsin is provided,
Comprising steps of
(1) the culture host cell obtains culture；With
(2) people's anionic trypsin mutant is separated from culture.
In another aspect of this invention, the purposes of people's anionic trypsin mutant is provided, for digesting egg
White matter or denatured protein, or it is used for cell cultivation process.
In another aspect of this invention, a kind of method of people's anionic trypsin stability improving recombination, institute are provided
The method of stating includes: to sport Ala and/or the 122nd by Ser for the 121st of people's anionic trypsin to be sported by Arg
Leu。
It in a preferred embodiment, further include that Cys and/or are sported by Ser by the 139th of people's anionic trypsin
206 sport Cys by Ser.
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, four kinds of trypsase primary sequence compare.Ht1 is people's cationic trypsase；Ht2 is people's anion
Type trypsase；Bt1 is bovine trypsin (SEQ ID NO:3)；Pt1 is Porcine trypsin (SEQ ID NO:4).
Fig. 2, mhTg2-S139C-S206C protein electrophoresis expression identification (a, b) and Western blot result (c).mhTg2
Indicate mutant human anionic trypsin.
(a-b) S139C-S206C protein electrophoresis expression identification result；(a) in, lane 1,3,5:S139C-S206C-
Before JM109 (DE3) induction；After lane 2,4,6:S139C-S206C-JM109 (DE3) induction；Lane 7,9,11:S139C-
Before S206C-Rosetta (DE3) induction；After lane 8,10,12:S139C-S206C-Rosetta (DE3) induction；(b) in,
Lane 1,3:37 DEG C of supernatant；Lane 2,4:37 DEG C of precipitating；Lane 5:25 DEG C supernatant；Lane 6:25 DEG C precipitating；Lane 7:15
DEG C supernatant；Lane 8:15 DEG C precipitating.
(c) S139C-S206C Western blot exercising result；Lane 9,12: after induction；Lane 10,13: supernatant；
Lane 11,14 is precipitated；M:Maker.
Fig. 3, S139C, S139C-S206C-R122L expression identification result.
Lane 1:S139C, before induction；After lane 2-4:S139C induction；Lane 5:S139C-S206C-R122L induction
Before；After lane 6-12S139C-S206C-R122L induction；M:Marker.
The elution curve and electrophoretogram of Fig. 4, S139C and S139C-S206C-R122L.
(a) elution curve of S139C.
(b) elution curve of S139C-S206C-R122L.
(c) the purifying electrophoretogram of S139C and S139C-S206C-R122L, lane1:S139C purify electrophoretogram；Lane 2:
S139C-S206C-R122L purifies electrophoretogram；M:Maker.
Fig. 5, S139C-S206C-S121A, S121A-R122L, S121A, S139C-S206C-S121A-R122L expression mirror
Determine result.
(a) before lane 1:S139C-S206C-S121A induction；After lane 2-4:S139C-S206C-S121A induction；
After lane 5:S121A-R122L induction；After lane6-12S139C-S206C-S121A-R122L induction；M:Maker；
(b) before lane 1:S121A induction；After lane 2-9:S121A induction；M:Maker.
Fig. 6, elution curve and Purification.
The purifying electricity of S139C-S206C-S121A, S139C-S206C-S121A-R122L, S121A-R122L and S121A
Swimming figure: lane1:S139C-S206C-S121A；Lane2:S139C-S206C-S121A-R122L；Lane3:S121A-R122L；
Lane4:S121A；(b) S139C-S206C-S121A elution curve；(c) S139C-S206C-S121A-R122L elution curve；
(d) S121A-R122L elution curve；(e) S121A elution curve.
The pH stability of Fig. 7, S139C and S139C-S206C-R122L.
The pH of Fig. 8, S121A, S139C-S206C-S121A, S139C-S206C-S121A-R122L and S121A-R122L
Stability.
The temperature stability of Fig. 9, S139C and S139C-S206C-R122L.
(1): S139C；(2): S139C-S206C-R122L.
Figure 10, S121A (1), S139C-S206C-S121A (2), S139C-S206C-S121A-R122L (3) and S121A-
The temperature stability of R122L (4).
Figure 11, hT1, hT2S121A；2:S139C-S206C-S121A；3:S139C-S206C-S121A-R122L；4:
S121A-R122L temperature stability compares (60 degree, 12h).
Figure 12, hT2, R122L, mhT2-S139C, mhT2-S139C-S206C-R122L HPLC result.
A:S139C-S206C-R122L；B:R122L；C:S139C；D:hT2WT.
Figure 13, hT2, R122L, S121A, S121A-R122L, S121A-S139C-S206C and S139C-S206C-
The HPLC of S121A-R122L temperature stability is detected.
A:S121A-R122L；B:S139C-S206C-S121A-R122L；C:R122L；D:S139C-S206C-S121A；
E:S121A；F:hT2 WT.
Specific embodiment
The present inventor pass through in-depth study, it has unexpectedly been found that some sites of people's anion tryptose and its to heat or pH
The stability of value is related, and the site is the 121st or the 122nd, obtains a kind of people's anion tryptose on this basis
Mutant, compared with wild type, the mutant stability with higher.Also, people's anion tryptose is in aforementioned mutation
On the basis of make effect even more ideal in conjunction with the selective mutation of the 139th or 206.
As used herein, unless otherwise stated, " mutant of people's anionic trypsin ", " people's trypsase
Mutant ", " mhTg2 ", " mutant of mutant human anionic trypsin ", " saltant type hTg2 " be used interchangeably, be
Refer to and correspond to wild type human anionic trypsin (such as SEQ ID NO:1), the 121st sports Ala and/or the 139th by Ser
Position sports Cys by Ser；It more preferably further include corresponding to SEQ ID NO:1 the 206th to sport Cys or the 122nd by Ser
The albumen that Leu is constituted is sported by Arg.
If desired the people's anionic trypsin for indicating wild type, will be denoted as " wild type human anion tryptose
Enzyme ", " hTg2 " or " wild-type protein ", amino acid sequence are SEQ ID NO:1.
As used herein, described " people's anionic trypsin (or mutant) activity " is determined with the unit of activity of enzyme
Justice.One unit of activity (1U) of enzyme is defined as 25 DEG C, under the conditions of pH7.6, reaction system 3.0ml (1cm optical path), per minute
Enzymatic hydrolysis N- Benzoyl-L-arginine ethyl ester (N-benzoyl-L-arginine ethyl ester, BAEE) makes the suction under 253nm
Receipts value increases 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 people's anionic trypsin mutant " refers to people's anionic trypsin mutant base
Without natural relative other albumen, lipid, carbohydrate or other materials in sheet.Those skilled in the art can use standard
Purified technology of protein Purification of Human anionic trypsin mutant.Substantially pure albumen is in non-reducing polyacrylamide gel
It is upper to generate single master tape.
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.
The invention also includes segment, the derivative and analogue of people's anionic trypsin mutant.Such as this paper institute
With term " segment ", " derivative " and " analog " refers to that being kept substantially natural human anionic trypsin of the invention dashes forward
The identical biological function of variant or active albumen.Protein fragments of the invention, derivative or the like, which can be (i), one
The substituted albumen of a or multiple conservative or non-conservative amino acid residues (preferably conservative amino acid), and such take
The amino acid residue in generation, which can be, may not be by genetic code encoding, or (ii) is in one or more amino acid residues
Albumen with substituent group, or the albumen that (iii) additional amino acid sequence is fused to this protein sequence and is formed are (such as leading
Sequence or secretion sequence or for purifying the sequence of this albumen or proprotein sequence or fusion protein).According to the definition of this paper this
A little segments, derivative and analogue belong to scope known to those skilled in the art.However, people's anion pancreas egg
White enzyme mutant and its segment, derivative and analogue amino acid sequence in, correspond to wild type human anionic trypsin,
121st sports Ala and/or the 139th by Ser and sports Cys by Ser；More preferably correspond to SEQ ID NO:1 the 206th
Position sports Cys or the 122nd by Ser and sports Leu by Arg.
In the present invention, term " people's anionic trypsin mutant " further includes (but being not limited to): several are (usually
Be 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
Within, more preferably it is within 5) amino acid.For example, in the art, being replaced with amino acid similar in performance
When, do not usually change the function of protein.For another example, usual in C-terminal and/or the one or several amino acid of N-terminal addition
The function of protein will not be changed.The term further includes that the active fragment of people's anionic trypsin mutant and activity derive
Object.But in these variant forms, correspond to wild type human anionic trypsin, the 121st by Ser sport Ala and/
Or the 139th sport Cys by Ser；More preferably correspond to SEQ ID NO:1 the 206th and sports Cys or the 122nd by Ser
Leu is sported by Arg.
The present invention also provides coding the present inventor's anionic trypsin mutant or its conservative variation's albumen it is more
Nucleotide 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 the maturation protein of the mutant include: a coded sequence for encoding mature albumen；It is mature
The coded sequence of albumen and various additional coding sequences；The coded sequence (and optional additional coding sequence) of maturation protein 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.
People's anionic trypsin mutant nucleotide full length sequence or its segment of the invention can usually use PCR amplification
Method, recombination method or artificial synthesized method obtain.It, can disclosed related nucleotides sequence according to the present invention for PCR amplification method
Column, especially open reading frame sequence carry out design primer, and with the commercially available library cDNA or by well known by persons skilled in the art normal
The library cDNA prepared by rule method expands as template and obtains related sequence.When sequence is longer, it is often necessary to carry out twice or
Then the segment that each time amplifies is stitched together by multiple 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 of the invention or people's anion pancreas
The genetically engineered host cell of protease mutant coded sequence, and albumen of the present invention is generated through recombinant technique
Method.
By the recombinant dna technology of routine (Science, 1984；224:1431), using polynucleotide of the invention
Sequence come express or produce recombination people's anionic trypsin mutant.In general there are following steps:
(1) polynucleotides (or variant) of encoding human anionic trypsin mutant of the invention, or with containing
The recombinant expression carrier of the polynucleotides 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, people's anionic trypsin mutant polynucleotide sequence be can be plugged into recombinant expression carrier.Art
Language " recombinant expression carrier " refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammal
Cell virus or other carriers.As long as any plasmid and carrier can be used in short, can replicate and stablize in host.Table
An important feature up to carrier 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 coding DNA of anionic trypsin mutant containing people sequence
The expression vector of column and suitable transcription/translation control signal.These methods include recombinant DNA technology in vi, DNA synthesis skill
Art, In vivo recombination technology etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, with guidance
MRNA synthesis.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, people's anionic trypsin mutant is produced by genetic engineering means, than
People's anionic trypsin mutant as described in using any suitable genetic engineering bacterium production, separates people's anion
Trypsin 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.
As preferred embodiment of the invention, the mutant is containing S121A or R122L or their complex mutation
Body, the complex mutation body containing S139C-S206C Yu S121A and/or R122L, these people's anionic trypsin mutants tool
There is good stability, show as that temperature stability is ideal, and pH stability is ideal, and bioactivity is high.Therefore, should
Mutant has important value for the practical application for widening trypsase.
HPLC analysis is carried out to mutant of the invention, obtains the single β-trypsin of peak type, do not contain or it is less containing
α-the trypsin of degradation.
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.
The expression and identification of embodiment 1, mutant S139C-S206C
1, point mutation designs
On the basis of wild type human anionic trypsin sequence, a variety of point mutation are designed, mutant is as follows:
(hT2-) S139C mutant: on the basis of SEQ ID NO:1 (ht2), the 139th sports C by S；
(hT2-) S139C-S206C mutant: on the basis of SEQ ID NO:1 (ht2), the 139th sports C by S, the
206 sport C by S；
(hT2-) S139C-S206C-R122L mutant: on the basis of SEQ ID NO:1 (ht2), the 139th is mutated by S
For C, the 206th sports C by S, and the 122nd sports L by R；
(hT2-) S121A mutant: on the basis of SEQ ID NO:1 (ht2), the 121st sports A by S；
(hT2-) R122L mutant: on the basis of SEQ ID NO:1 (ht2), the 122nd sports L by R；
(hT2-) S139C-S206C-S121A mutant: on the basis of SEQ ID NO:1 (ht2), the 139th is mutated by S
For C, the 206th sports C by S, and the 121st sports A by S；
(hT2-) S121A-R122L mutant: on the basis of SEQ ID NO:1 (ht2), the 121st sports A by S, the
122 sport L by R；
(hT2-) S139C-S206C-S121A-R122L mutant: on the basis of SEQ ID NO:1 (ht2), the 139th
C is sported by S, the 206th sports C by S, and the 121st sports A by S, and the 122nd sports L by R.
HT1-C139S-C206S mutant: on the basis of SEQ ID NO:2 (ht1), the 139th sports S by C.
Sheng Gong Bioisystech Co., Ltd is transferred to prepare the coded sequence of the polypeptide of above-mentioned point mutation (through Escherichia coli preference
Codon optimization), be inserted into the site NdeI/Hind III of pET-32a plasmid, after sequencing is correct, point mutation can be obtained
Recombinant plasmid, for recombinantly expressing.
2, the expression identification of recombinant plasmid
By the recombinant plasmid of the coded sequence of the people's anionic trypsin for inserting point mutation of aforementioned preparation, it is transferred to
In E.coli BL21 (DE3) competent cell.It is put into after coated plate in 37 DEG C of constant incubators, is inverted culture 12-16h.It chooses respectively
Single colonie is taken to be placed in 30ml shaking flask, to OD600When=0.5-0.6, IPTG is added and induces, after 37 DEG C of culture 4h, carries out SDS-
PAGE identification.As a result see Fig. 2.
3, ultrasonication
By the bacterium solution after Fiber differentiation, it is centrifuged 20min in 3000rpm, thallus is collected, with 50mmol/L Tris-HCl pH
8.0 buffers sufficiently suspend thallus.The bacterium solution to have suspended is placed in Ultrasonic Cell Disruptor, work 5s, is spaced 5s, ultrasonic power
200W is recycled 99 times.By broken bacterium solution in 12000rpm, 4 DEG C of centrifugation 10min, supernatant precipitating is separated.Take supernatant heavy respectively
It forms sediment and carries out SDS-PAGE identification.
4, the experimental procedure of Western blot
Protein electrophoresis glue is placed on pvdf membrane, is covered with filter paper, is placed in electric turn trough and shifts 1-2 hours (1mA-2mA/
Cm2,10%gel), the confining liquid containing 5% skim milk is added after transfer, after incubation at room temperature 1 hour, after dilution is added
Primary antibody, be incubated at room temperature 1 hour, after washing off primary antibody, be incubated for 2 hours according to the secondary antibody that added primary antibody is added after dilution, by
ECL colour developing developing fixing, observes result.As a result see Fig. 3.
5, result
Plasmid comprising encoding mutant body S139C-S206C is converted to e. coli jm109 (DE3) and Rosetta
(DE3) it expresses in bacterium and carries out expression identification, as shown in Fig. 2 (a), have no apparent band of expression.But it after passing through induction and lures
From the point of view of the swimming lane of leading is compared relatively, there is apparent degradation band in 14kDa or less.
Will in S139C-S206C gene cloning to pET-32a plasmid, select respectively 15 DEG C, 25 DEG C, under the conditions of 37 DEG C of temperature
Inducing expression identification is carried out, such as Fig. 2 (c).It was found that 14kDa or less has an apparent expression in the precipitating at 15 DEG C and 25 DEG C
Band, but be not inconsistent at the 29kDa of destination protein position.In 37 DEG C, there is a deeper band in the precipitating of 29kDa or more, but
It is not precipitated largely in follow-up test.After carried out Western Blot identification (Fig. 2 (b)).The result shows that 25
A small amount of expression can be seen in DEG C precipitating, and has apparent small molecule band, thus infers, it may be possible to S139C-
S206C is extremely unstable during expression, to can degrade.
The expression of embodiment 2, mutant S139C, S139C-S206C-R122L
Building and expression identification are carried out according to the method for embodiment 1.As a result see Fig. 3.
By can be seen that S139C mutant and S139C-S206C-R122L after being compared the swimming lane of induction front and back
Mutant has an apparent band of expression in 29kDa or so, and expression quantity about accounts for 60% of total protein concentration or more.
The purifying of embodiment 3, S139C and S139C-S206C-R122L
1, the washing and renaturation of inclusion body
The precipitating that broken bacterium obtains is resuspended in containing 0.5%Triton X-100 (v:v), 20mmol/L Tris-
In the solution of HCl pH 8.0,1mmol/L EDTA, after stirring 1h at room temperature, supernatant is removed in 12000rpm, 4 DEG C of centrifugation 10min,
It collects to be resuspended in again in 8.0 solution of 20mmol/L Tris-HCl pH after precipitating and repeat the above steps twice.It is finally that washing is dry
Net inclusion body 8M Urea solution dissolves, and is diluted renaturation.
2, purifying pretreatment
Enzymatic activity and activation situation in renaturation solution are detected by enzyme activity determination and SDS-PAGE.By renaturation solution 12000rpm from
Heart removal precipitating, supernatant dialyse to 1mmol/L HCl with 1:10 (v:v).It is primary that dialysis 6h changes external solution, replaces 4 times altogether.It will be saturating
Renaturation solution after analysis carries out next step purifying.
3, CM-FF ion-exchange chromatography
Using 2 × 15cm chromatographic column, it is previously added the water of 1/10 column volume, CM-FF is adsorbed resin water by closing outlet
The solution for suspending into 50% (v:v) is fitted into chromatographic column using glass bar drainage.It is used after being rinsed using the water of 2 times of column volumes
0.5M NaOH solution slow processes 1-2h.It is rinsed with the water of 5 times of column volumes to neutrality, then with 2 cylinder of 1M NaCl slow processes
Product.10 column volumes are balanced with 5.0 buffer of 20mmol/L NaAc-HAc pH, renaturation solution is adjusted to 200Mmol/L NaAc
PH 5.0, connection UV detector start loading.With 5.0 buffer of 20mmol/L NaAc-HAc pH balance 2 after end of the sample
Column volume.It is buffered when elution using the 20mmol/L NaAc-HAc pH 5.0 of 10 times of column volume NaCl Han 0~500mmol/L
The elution of liquid continuous gradient.Purpose peak, the every pipe OD280 of sequentially determining and enzyme activity are collected according to UV detector numerical value change, and is counted
Calculate the rate of recovery of albumen.
4, the detection of people's trypsase vigor
Using N- Benzoyl-L-arginine ethyl ester (N-benzoyl-L-arginine ethyl in the present invention
Ester, BAEE) as substrate detection people's trypsase vigor.Since people's trypsase can specific for hydrolysis arginine one of carbon tip
Peptide bond, therefore N- Benzoyl-L-arginine ethyl ester (BAEE) can be degraded to N- Benzoyl-L-arginine (benzoyl-L-
Arginine, BA).Under 253nm wavelength, the absorption value of N- Benzoyl-L-arginine ethyl ester (BAEE) is far smaller than its degradation
Object N- Benzoyl-L-arginine (benzoyl-L-arginine, BA).Under certain conditions, with catalysis reaction progress,
Product BA gradually increases, and the ultraviolet absorption value of system gradually increases, finally with the variable quantity △ of ultraviolet absorption value under 253nm
A253nm calculates the activity of people's trypsase, increases △ A253nm by 0.001 one BAEE unit of enzyme amount.Enzyme activity
Property scale merit be 1 USP Unit be equal to 3 BAEE Units.When preparing BAEE substrate, the buffer used is
25mmol/L Tris-HCl pH7.6 contains 0.1mol/L NaCl and 0.01mol/L CaCl2.The working concentration of substrate is
25mmol/L, surveying temperature living is 25 DEG C.
5, result
As a result as shown in Figure 4.By CM-FF ion-exchange chromatography, more pure albumen is obtained, in the process of elution
Middle discovery, mutant S139C can elute next small peak near 100mmol/L NaCl, substantially without enzyme activity, it may be possible to degrade
The small peptide got off.Then appearance is relatively simple by mutant S139C-S206C-R122L, and eluting peak relatively early stage binding ability is slightly
It is weaker than mutant S139C.
Identified by protein electrophoresis, the purity of two kinds of albumen has all reached target, measure after purification S139C and
The ratio of S139C-S206C-R122L living is respectively 16018.2U/mg and 10332.1U/mg.
Embodiment 4, mutant S139C-S206C-S121A, S121A-R122L, S121A, S139C-S206C-S121A-
The expression and purifying of R122L
The weight of mutant S139C-S206C-S121A, S121A-R122L, S121A, S139C-S206C-S121A-R122L
Group expression and identification method are carried out according to embodiment 1.
Recombinant plasmid transformed is entered in e. coli bl21 (DE3) expression bacterium, expression identification is carried out, as a result such as Fig. 5 institute
Show, near 29kDa, mutant S139C-S206C-S121A, S121A-R122L, S139C-S206C-S121A-R122L and
S121A is expressed.It is unable to get the S139C-S206C mutant completely expressed in embodiment 1, but adds here
After entering S121A mutation, the expression of S139C-S206C-S121A has been obtained, it is seen then that S121A plays stable protein expression
The effect of conspicuousness.
Purification process is carried out referring to embodiment 3, as a result sees Fig. 6.
As shown in Fig. 6 (a), by after purification, four kinds of mutant have obtained purer albumen.Come from elution curve
It sees, S139C-S206C-S121A-R122L binding ability is slightly strong, and such as Fig. 6 (c), other four kinds of protein binding capacities are all close,
S121A such as Fig. 6 (d) has a foreign protein peak in front.
Mutant S139C-S206C-S121A is after purification than living for 11000U/mg, S139C-S206C-S121A-R122L
It is after purification 13500U/mg, S121A-R122L ratio work than work be the ratio work of 11500U/mg, S121A is after purification 10000U/
mg。
The pH stability of embodiment 5, ht2 disulfide bond S139C-S206C series mutants
In the present embodiment, ht2 series mutants S139C and S139C-S206C-R122L and wild type hT1 and hT1 are carried out
The comparison of the pH stability of mutant.
Enzyme solution is respectively placed in 3~11 buffer of pH, protein concentration is controlled in 0.5mg/ml, the water-bath 2h at 25 DEG C
Measurement activity, buffer are followed successively by 50mmol/L NaAc-HAc (pH 3~6) afterwards, Tris-HCl (pH 7~8), Gly-NaOH
(pH 9~11), all equal 25 DEG C of preparations of buffer.Using the enzyme solution initial activity before water-bath as 100%, calculate under corresponding pH
Remnant enzyme activity.
The pH of mutant S139C, S139C-S206C-R122L, R122L and hT2, hT1 and hT1-C139S-C206S stablize
The comparison result of property is as shown in Figure 7.It is contemplated respectively at various ph values, the situation of change of enzyme activity after 25 DEG C of water-bath 2h.hT1
Good stability is shown in the range of pH3-11, remnant enzyme activity is 80% or more.In the pole pH3-4 and 10-11
It is stablized, and will not influence enzyme activity substantially, drops to 80% in pH5 or so enzyme activity.In contrast, hT2 is only shown in pH3
Certain stability, within the scope of pH4-11, remnant enzyme activity is no more than 50%, in pH5 and pH9 most unstable, remaining
Living only 20%.Mutant hT2-R122L improves the pH stability of hT2, especially alkali resistance to a certain extent, in pH7-
20% remnant enzyme activity is improved in the range of 11 than hT2 wild type.The pH of mutant S139C and hT2 stablize similar temperament, right
The stability of soda acid slightly improves.HT1-C139S-C206S this to disulfide bond missing mutant compared with hT1 wild type, pH
Stability decline is obvious, and in the range of pH4 and 6-11, remnant enzyme activity is below 50%.S139C-S206C-R122L mutant
Good stability is shown, in addition to pH9, remnant enzyme activity is 50% or more under other pH, and in pH3-4 and pH11
Neighbouring enzyme activity is not lost substantially.PH stability greatly improves compared with hT2 wild type.
Embodiment 6, the pH stability of people's anionic trypsase S121A series mutants compare
In the present embodiment, ht2 series mutants S121A, S139C-S206C-S121A, S139C-S206C- are carried out
S121A-R122L and S121A-R122L is compared with the pH stability of wild type hT1 and ht2.
Enzyme solution is respectively placed in 3~11 buffer of pH, protein concentration is controlled in 0.5mg/ml, the water-bath 2h at 25 DEG C
Measurement activity, buffer are followed successively by 50mmol/L NaAc-HAc (pH 3~6) afterwards, Tris-HCl (pH 7~8), Gly-NaOH
(pH 9~11), all equal 25 DEG C of preparations of buffer.Using the enzyme solution initial activity before water-bath as 100%, the corresponding pH of computational item
Under remnant enzyme activity.
The pH stability of S121A series mutants as shown in figure 8, be contemplated it at various ph values respectively, 25 DEG C of water-baths
The situation of change of enzyme activity after 2h.Mutant S139C-S206C-S121A, S121A-R122L, S121A compared with hT2 wild type,
It is about the same in each pH stability inferior.S121A-R122L improves 10% or so remnant enzyme activity in the range of pH6-11.
But hT2-S139C-S206C-S121A-R122L but significantly improves pH stability, in the range of pH3-11 from the point of view of, remove
Other than pH9, remnant enzyme activity is higher than hT1 wild type near pH5 and pH7 80% or more, but in pH9, remaining
Enzyme activity but has decreased to 50% hereinafter, this is also the general character of hT2 and its mutant.
Embodiment 7, the temperature stability of people's trypsase disulfide bond mutant compare
In the present embodiment, carry out S139C's and S139C-S206C-R122L and wild type hT1 and hT1 mutant S121A
Stability compares.
The enzyme solution for taking 0.5mg/ml after purification, be respectively placed in 4 DEG C, 25 DEG C, 37 DEG C, 40 DEG C, 50 DEG C, incubate in 60 DEG C of water-baths
Enzyme activity is measured by sampling every 30min in 12h, residual enzyme of the initial activity of enzyme solution as 100% calculating at each temperature using before water-bath
It is living.
See Fig. 9, mutant S139C and S129C-S206C-R122L are contemplated respectively, (albumen is whole in 5mmol/L HCl
Concentration 1mg/ml) it is placed in the situation of change of enzymatic activity in 12h at 4~60 DEG C.Two kinds of mutant 4 DEG C, 15 DEG C, 25 DEG C, 37 DEG C,
At 40 DEG C, vigor is relatively stable in 12h, and remnant enzyme activity is 80% or more.At 50 DEG C, the decline of mutant S139C activity is bright
Aobvious, vigor only has the 20% of initial enzyme activity after vigor has declined 50%, 12h in 4h, and S139C-S206C-R122L is residual in contrast
It is 82% after remaining enzyme activity 12h.At 60 DEG C, mutant S139C activity declines more rapid, and vigor just drops to after 2h
Vigor almost exhausts after 20%, 4h.And S139C-S206C-R122L is after 60 DEG C of placement 12h, can still retain 60% with
On activity.
The comparison of embodiment 8, people's trypsase S121A series mutants temperature stability
Method is the same as embodiment 7.As a result such as Figure 10, in several S121A mutant at different temperatures 5mmol/L HCl
Activity change situation in (final concentration of protein 1mg/ml) 12h.At 25 DEG C hereinafter, in 12h, four kinds of mutant activity situations are basic
Stablize.At 37 DEG C to 50 DEG C, 80% residual activity can be also remained with.60 DEG C, mutant S139C-S206C- after 12h
S121A-R122L, still remains 70% or more vigor, but S121A, S139C-S206C-S121A, S121A-R122L this
Three kinds of mutant, vigor all have dropped 50% or so.It so this had not only included the mutation of disulfide bond, but also include from enzyme site
The mutant S139C-S206C-S121A-R122L of mutation, stability are fine.
Two groups of mutant and wild type are placed at 60 DEG C by detection together, in 1mmol/L HCl, remnant enzyme activity after 12h,
As a result as shown in figure 11, at 60 DEG C, after 12h, the activity decline of ht2 wild type complete deactivation, hT2-S139C faster also exists
Complete deactivation after 12h.HT2-R122L mutant final residual enzyme activity is 50%.HT1 remnant enzyme activity be 60%, S121A,
The final enzyme activity of S139C-S206C-R122L, S139C-S206C-S121A mutant is suitable therewith, but activity decline is slower.
But S139C-S206C-S121A-R122L heat resistance is fabulous, remnant enzyme activity is still 70% or more.
The HPLC of embodiment 9, people's trypsase and its series mutants analyzes result
HPLC detects people's trypsase, the method is as follows:
Mobile phase
Mobile phase A: contain 0.1%H3PO4(85%) aqueous solution.
Mobile phase B: contain 0.1%H3PO4(85%) acetonitrile.
Using preceding with 0.22 μ l membrane filtration degerming, ultrasonic degassing.
Chromatographic program: referring to United States Pharmacopeia " Enzymes Used As Ancillary Material in 2013
To the regulation of recombined human trypsase HPLC measuring method in Pharmaceutical Manufacturing ".Using C18 column
(EF-C18 (H) 4.6 × 250mm, 3 μm)。
HPLC mobile phase elution program:
Sample volume is 1-20 μ l, and protein content is no less than 50 μ g in each loading.Flow velocity is 1.0ml/min.Column temperature is 40 DEG C.
Detection wavelength is 280nm.
Using the trypsase of various configuration in the standard analysis trypsase of HPLC trypsase United States Pharmacopeia, when separation
Between be 30min, wherein different according to trypsase configuration, the retention time of α-trypsin and β-trypsin are 12-17 minutes.
The HCl that final concentration of 1mol/L is separately added into before loading carries out acidification to sample.
As can be seen that hT2 appearance time is 13.1min from Figure 12 (D), it is there are two small peak before β-trypsin
α-trypsin.As shown in Figure 12 (C), S139C appearance time is essentially identical with hT2 but main peak is not single enough, it may be possible to sample
Not pure enough or conformation is not single enough.R122L appearance time is later relatively simple for 15.8min peak type, sees Figure 12 (B).From figure
The visible S139C-S206C-R122L appearance time of 12 (A) is later, is 16.3min, but its main peak is relatively simple, and does not have
α-trypsin exists.
Such as Figure 13, found by comparing the HPLC testing result of several mutant, the mutant appearance with the site R122L
Time can all become late.And after being added to disulfide bond, the site R122L and S121A, appearance time can also be delayed.But all in all
Peak type is all relatively simple, can all obtain purer β-trypsin.Without the mutation in the mutational site S121A R122L
Body, as S139C-S206C-S121A and S121A can have α-trypsin, but hT2 wild type becomes apparent, in α-
More degradation bands are also had before the position trypsin.
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.
Claims (7)
1. the mutant of people's anionic trypsin, which is characterized in that the amino acid sequence of the mutant corresponds to SEQ ID
NO:1, the 121st by Ser sport Ala, the 139th by Ser sport Cys, the 206th Cys and the 122nd are sported by Ser
Position sports Leu by Arg.
2. isolated polynucleotides, which is characterized in that mutant described in the polynucleotide encoding claim 1.
3. a kind of carrier, which is characterized in that it contains polynucleotides as claimed in claim 2.
4. a kind of genetically engineered host cell, which is characterized in that it contains carrier or genome as claimed in claim 3
In be integrated with polynucleotides as claimed in claim 2.
5. a kind of method for the mutant for producing people's anionic trypsin described in claim 1, which is characterized in that including step
It is rapid:
(1) host cell as claimed in claim 4 is cultivated, culture is obtained；With
(2) people's anionic trypsin mutant described in claim 1 is separated from culture.
6. the purposes of people's anionic trypsin mutant described in claim 1 is used for enzymolysis protein matter or albuminate
Matter, or it is used for cell cultivation process.
7. a kind of method for the people's anionic trypsin stability for improving recombination, which is characterized in that the described method includes: by people
The 121st of anionic trypsin by Ser sport Ala, the 122nd by Arg sport Leu, the 206th be mutated by Ser
Leu is sported by Arg for Cys and the 122nd.
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| R122L突变提高重组人阴离子型胰蛋白酶的稳定性的研究;马强等;《中国生化药物杂志》;20141231;第34卷(第2期);第61-64页 |
| 重组人源阳离子型胰蛋白酶研究;李强;《中国优秀硕士学位论文全文数据库 基础科技辑》;20130615(第06期);第39页第1段，第42页倒数第1段-第1段，第45页第1段 |
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