CN106337043A - Human trypsin mutant with high stability - Google Patents
Human trypsin mutant with high stability Download PDFInfo
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- CN106337043A CN106337043A CN201510418411.7A CN201510418411A CN106337043A CN 106337043 A CN106337043 A CN 106337043A CN 201510418411 A CN201510418411 A CN 201510418411A CN 106337043 A CN106337043 A CN 106337043A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21004—Trypsin (3.4.21.4)
Abstract
The invention relates to a human trypsin mutant with high stability. It is found in the invention that a plurality of sites of human anionic trypsin are related to stability of the human anionic trypsin to heat or pH value; a site involved in the invention is site 121 or site 122; a human anionic trypsin mutant is obtained on the basis of the site; and compared with wild human anionic trypsin, the mutant has high stability. Moreover, the mutant is combined with dual-mutation of the site 139 and the site 206 of human anionic trypsin, so enzyme stability and activity are more ideal.
Description
Technical field
The invention belongs to biological technical field；More particularly it relates to people's Trypsin of high stability
Enzyme mutant.
Background technology
Since extracorporeal recombination is applied so far from the maturation seventies, substantial amounts of vivo protein is by recombinant clone.
Recombiant protein, compared to the albumen extracting in animal body, has several obvious greatly advantages: be not related to first move
Thing is originated, non-animal derived property production process；Secondly, background understands, purity is high, suitable reconstituted drug life
Produce, serum-free culture etc..
, in clinical immunotherapy of tumors, the process such as tissue culture, production of vaccine for man is equal for trypsin
Indispensable, now use trypsin to be animal derived, have the probability of virus pollution, thus leading to people to store
Ill propagation altogether, the recombinant trypsin using non-animal derived property is the basic scheme solving this problem.
In 2014, American Pharmacopeia, for the recombinant trypsin being applied to during bio-pharmaceuticals, was given
Recombinant trypsin up-to-date examination criteria.In skin tissue cell incubation, non-animal derived property
Favoring of being increasingly subject to of product.Went et al. is respectively with animal derived trypsin and the weight of extraction
Corneocyte in group trypsinization epidermal area.Show skin histology after recombinant trypsin digestion
The growing state of cell will be substantially better than the cell after animal derived pancreatin is processed.
To sum up, this area needs the trypsin of large-scale production people source.But natural people's Trypsin
The activity of enzyme enzyme is low, stability is poor, greatly limit its popularization and application.Therefore, this area in the urgent need to
Find the effective means improving its stability.
Content of the invention
It is an object of the invention to provide a kind of people's trypsin mutant of high stability.
In a first aspect of the present invention, provide the mutant of people's anionic trypsin, described mutant
Aminoacid sequence corresponds to seq id no:1, and the 121st sports ala and/or the 139th by ser
Cys is sported by ser.
In a preference, the aminoacid sequence of described mutant also includes: corresponding to seq id no:
1, the 206th sports cys by ser；Or the 122nd sport leu by arg；And, it is described
Mutant be not aminoacid sequence such as seq id no:1 aminoacid sequence and the 139th be mutated by ser
For cys, the 206th mutant being sported cys by ser.
In another preference, described mutant is:
As the aminoacid sequence of seq id no:1, but wherein the 139th by ser sport cys,
206 by ser sport cys, the 121st ala the 122nd is sported by ser and is sported by arg
Leu (abbreviation s139c-s206c-s121a-r122l)；Or
As the aminoacid sequence of seq id no:1, but wherein the 139th by ser sport cys,
206 sport cys and the 122nd by ser and sport leu (referred to as by arg
s139c-s206c-r122l)；Or
As the aminoacid sequence of seq id no:1, but wherein the 139th by ser sport cys,
206 sport cys and the 121st by ser and sport ala (referred to as by ser
s139c-s206c-s121a)；Or
As the aminoacid sequence of seq id no:1, but wherein the 121st sports ala and by ser
122 sport leu (abbreviation s121a-r122l) by arg.
In another preference, in ph3-11,25 DEG C preserve 2h to s139c-s206c-s121a-r122l,
The vigor of enzyme is held in more than 80%；Ph3,60 DEG C preserve 12h remnant enzyme activity may remain in 70% with
On；Its high temperature resistance and ph toleration are splendid.
In another aspect of this invention, detached polynucleotide are provided, described in described polynucleotide encoding
Mutant.
In another aspect of this invention, provide a kind of carrier, it contains described polynucleotide.
In another aspect of this invention, provide a kind of genetically engineered host cell, it contains described load
It is integrated with described polynucleotide in body, or genome.
In another aspect of this invention, provide and a kind of produce the described mutant of people's anionic trypsin
Method, including step:
(1) cultivate described host cell, obtain culture；With
(2) separate described people's anionic trypsin mutant from culture.
In another aspect of this invention, the purposes of described people's anionic trypsin mutant is provided, is used for
Enzymolysis protein matter or denatured protein, or it is used for cell cultivation process.
In another aspect of this invention, provide a kind of side of the people's anionic trypsin stability improving restructuring
Method, methods described includes: by the 121st of people's anionic trypsin by ser sport ala and/or
122nd sports leu by arg.
In a preference, also include being sported the 139th of people's anionic trypsin by ser
Cys and/or the 206th sports cys by ser.
The other side of the present invention, due to this disclosure, is aobvious to those skilled in the art
And be clear to.
Brief description
Fig. 1, four kinds of tryptic primary sequences compare.Ht1 is people's cationic trypsin；ht2
Behaviour anionic trypsin；Bt1 is bovine trypsin (seq id no:3)；Pt1 is Porcine trypsin
(seq id no:4).
Fig. 2, mhtg2-s139c-s206c protein electrophoresises expression identification (a, b) and western blot result
(c).Mhtg2 represents mutant human anionic trypsin.
(a-b) s139c-s206c protein electrophoresises expression identification result；In (a), lane 1,3,5:
Before s139c-s206c-jm109 (de3) induction；Lane 2,4,6:s139c-s206c-jm109 (de3)
After induction；Lane 7,9, before 11:s139c-s206c-rosetta (de3) induction；Lane 8,10,12:
After s139c-s206c-rosetta (de3) induction；In (b), lane 1,3:37 DEG C of supernatant；Lane 2,4:
37 DEG C of precipitations；5:25 DEG C of supernatant of lane；6:25 DEG C of precipitation of lane；7:15 DEG C of supernatant of lane；Lane 8:
15 DEG C of precipitations.
(c) s139c-s206c western blot exercising result；Lane 9,12: after induction；lane 10、
13: supernatant；Lane 11,14 precipitates；M:maker.
Fig. 3, s139c, s139c-s206c-r122l expression identification result.
Lane 1:s139c, before induction；After lane 2-4:s139c induction；Lane 5:
Before s139c-s206c-r122l induction；After lane 6-12s139c-s206c-r122l induction；m:
marker.
The elution curve of Fig. 4, s139c and s139c-s206c-r122l and electrophoretogram.
The elution curve of (a) s139c.
The elution curve of (b) s139c-s206c-r122l.
The purification electrophoretogram of (c) s139c and s139c-s206c-r122l, lane1:s139c purification electrophoresis
Figure；Lane 2:s139c-s206c-r122l purification electrophoretogram；M:maker.
Fig. 5, s139c-s206c-s121a, s121a-r122l, s121a, s139c-s206c-s121a-
R122l expression identification result.
Before (a) lane 1:s139c-s206c-s121a induction；Lane 2-4:s139c-s206c-s121a
After induction；After lane 5:s121a-r122l induction；lane6-12s139c-s206c-s121a-r122l
After induction；M:maker；
Before (b) lane 1:s121a induction；After lane 2-9:s121a induction；m:maker.
Fig. 6, elution curve and Purification.
S139c-s206c-s121a, s139c-s206c-s121a-r122l, s121a-r122l and
The purification electrophoretogram of s121a: 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.
Fig. 8, s121a, s139c-s206c-s121a, s139c-s206c-s121a-r122l and
The ph stability of s121a-r122l.
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)
Temperature stability with s121a-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 ties
Really.
A:s139c-s206c-r122l；B:r122l；C:s139c；D:ht2wt.
Figure 13, ht2, r122l, s121a, s121a-r122l, s121a-s139c-s206c and
The hplc detection of s139c-s206c-s121a-r122l temperature stability.
A:s121a-r122l；B:s139c-s206c-s121a-r122l；C:r122l；D:
s139c-s206c-s121a；E:s121a；F:ht2 wt.
Specific embodiment
The present inventor is through in-depth study it has unexpectedly been found that some sites of people's anion Trypsin are right with it
The stability of heat or ph value is related, and described site is the 121st or the 122nd, obtains on this basis
One class people's anion Trypsin mutant, compared with wild type, described mutant has higher stablizing
Property.And, the choosing in conjunction with the 139th or 206 on the basis of aforementioned mutation of people's anion Trypsin
The mutation of selecting property makes effect even more ideal.
As used herein, unless otherwise indicated, described " mutant of people's anionic trypsin ",
" the tryptic mutant of people ", " mhtg2 ", " mutant of mutant human anionic trypsin ",
" saltant type htg2 " is used interchangeably, and refers to corresponding to wild type human anionic trypsin (as seq id
No:1), the 121st sports ala and/or the 139th by ser and sports cys by ser；More preferably
Also include sporting cys or the 122nd corresponding to seq id no:1 the 206th by ser and dashed forward by arg
It is changed into the albumen of leu composition.
If desired represent people's anionic trypsin of wild type, it will be denoted as " wild type human anion
Trypsin ", " htg2 " or " wild-type protein ", its aminoacid sequence is seq id no:1.
As used herein, described " people's anionic trypsin (or mutant) activity " is the vigor with enzyme
Unit is defining.One unit of activity (1u) of enzyme is defined as 25 DEG C, under the conditions of ph7.6, reaction system
3.0ml (1cm light path), enzymolysis n- benzoyl-l- arginine ethyl ester (n-benzoyl-l-arginine per minute
Ethyl ester, baee) make the absorption value increase by 0.001 under 253nm be defined as a baee unit.
As used herein, " detached " refers to that material is separated (if natural from its primal environment
Material, primal environment is natural surroundingses).As the polynucleotide under the native state in active somatic cell
Do not isolate and purify with albumen, but same polynucleotide or albumen are deposited as same from native state
Other materials in separately, then isolate and purify.
As used herein, " detached people's anionic trypsin mutant " refers to people's anionic trypsin
Mutant is substantially free of natural relative other albumen, lipid, saccharide or other materials.Ability
The technical staff in domain can use the purified technology of protein Purification of Human anionic trypsin mutant of standard.Base
In basis, pure albumen can produce single master tape in non-reducing polyacrylamide gel.
As used herein, " restructuring " refers to obtain the egg of (or a large amount of preparation) by genetic engineering means
In vain, engineering carrier or cell etc..
The albumen of the present invention can be recombiant protein, native protein, synthetic proteinses, preferably recombiant protein.This
The albumen of invention can be native purified product, or the product of chemosynthesis, or uses recombinant technique
Produce from protokaryon or eucaryon host (for example, antibacterial, yeast, higher plant, insecticide and mammalian cell)
Raw.
Present invention additionally comprises the fragment of described people's anionic trypsin mutant, derivant and analog.As
Used herein, term " fragment ", " derivant " and " analog " refers to be kept substantially the present invention
Natural human anionic trypsin mutant identical biological function or activity albumen.The present invention's
Protein fragments, derivant or the like can be that (i) has one or more conservative or non-conservative amino acid residue
(preferably conservative amino acid) substituted albumen, and such substituted amino acid residue can be
Can not be by genetic code encoding, or (ii) has substituted radical in one or more amino acid residues
Albumen, or the albumen that (iii) additional aminoacid sequence is fused to this protein sequence and is formed is (as targeting sequencing
Or secretion sequence or the sequence or the proprotein sequence that are used for this albumen of purification, or fusion protein).According to herein
These fragments of definition, derivant and analog belong to scope known to those skilled in the art.So
And, the aminoacid sequence of described people's anionic trypsin mutant and its fragment, derivant and analog
In row, corresponding to wild type human anionic trypsin, the 121st sports ala and/or by ser
139 sport cys by ser；More preferably sported by ser corresponding to seq id no:1 the 206th
Cys or the 122nd sports leu by arg.
In the present invention, term " people's anionic trypsin mutant " also includes (but being not limited to): if
Dry (usually 1-20, more preferably 1-10, also more preferably as 1-8,1-5,1-3 or
1-2) disappearance of aminoacid, insertion and/or replacement, and add in c end and/or n end or lack
One or several (usually 20 within, within preferably 10, more preferably within 5) amino
Acid.For example, in the art, when being replaced with similar nature or similar aminoacid, generally will not
Change the function of protein.Again such as, add one in c end and/or n end or several aminoacid leads to
Often also will not change the function of protein.This term also includes the activity of people's anionic trypsin mutant
Fragment and reactive derivative.But in these variant forms, corresponding to wild type human anion Trypsin
Enzyme, the 121st sports ala and/or the 139th by ser and sports cys by ser；More preferably corresponding
Sport cys or the 122nd in seq id no:1 the 206th by ser and leu is sported by arg.
Present invention also offers coding the present inventor's anionic trypsin mutant or its conservative variation's egg
White polynucleotide sequence.
The polynucleotide of the present invention can be dna form or rna form.Dna form include cdna,
The genome dna or dna of synthetic.Dna can be single-stranded or double-strand.Dna is permissible
It is coding strand or noncoding strand.
The polynucleotide encoding the maturation protein of described mutant include: the code sequence of an encoding mature albumen
Row；The coded sequence of maturation protein and various additional coding sequence；The coded sequence of maturation protein is (and optionally
Additional coding sequence) and non-coding sequence.
Term " polynucleotide of encoding proteins " can be including encode this albumen polynucleotide it is also possible to
It is the polynucleotide also including additional code and/or non-coding sequence.
The invention still further relates to the variant of above-mentioned polynucleotide, its coding and the present invention have identical aminoacid sequence
The fragment of the albumen of row or albumen, analogs and derivatives.The variant of this polynucleotide can be natural
The allelic variant occurring or the variant of non-natural generation.These nucleotide variants include replacing variation
Body, Deletion variants and insert variation.As known in the art, allelic variant is nucleoside more than
The alternative forms of acid, it is probably the replacement of one or more nucleotide, disappearance or insertion, but will not be from
Substantially change the function of the albumen of its coding.
People's anionic trypsin mutant nucleotide full length sequence of the present invention or its fragment generally can be used
The method of pcr TRAP, recombination method or synthetic obtains.For pcr TRAP, can be according to this
Bright disclosed relevant nucleotide sequence, especially open reading frame sequence designing primer, and with commercially available
Cdna storehouse or the cdna storehouse as prepared by conventional method well known by persons skilled in the art as template,
Expand and obtain relevant sequence.When sequence is longer it is often necessary to carry out twice or multiple pcr amplification, so
The fragment amplifying each time more afterwards is stitched together by proper order.
Once obtaining relevant sequence it is possible to obtain relevant sequence in large quantity with recombination method.This leads to
It is often to be cloned into carrier, then proceeds to cell, then the host cell after propagation by conventional method
Middle separation obtains relevant sequence.
Additionally, also relevant sequence can be synthesized with the method for synthetic, when especially fragment length is shorter.
Generally, by first synthesizing multiple small fragments, then it is attached obtaining the very long fragment of sequence again.
At present it is already possible to obtain by chemosynthesis encoding completely albumen of the present invention (or its fragment, or
Its derivant) dna sequence.Then this dna sequence can be introduced as known in the art various existing
Dna molecule (or as carrier) and cell in.Additionally, also mutation can be introduced this by chemosynthesis
In bright protein sequence.
The present invention also relates to comprising the carrier of the polynucleotide of the present invention, and the carrier with the present invention or people's the moon
The host cell that ion trypsin mutant coded sequence produces through genetic engineering, and through recombinant technique
The method producing albumen of the present invention.
By conventional restructuring dna technology (science, 1984；224:1431), the available present invention
Polynucleotide sequence is expressing or to produce people's anionic trypsin mutant of restructuring.In general have
Following steps:
(1). with the polynucleotide (or variant) of the encoding human anionic trypsin mutant of the present invention, or
With the conversion of the recombinant expression carrier containing this polynucleotide or suitable host cell of transduceing；
(2). the host cell of culture in suitable culture medium；
(3). separation, protein purification from culture medium or cell.
In the present invention, people's anionic trypsin mutant polynucleotide sequence can be plugged into recombinant expression carrier
In.Term " recombinant expression carrier " refer to bacterial plasmid well known in the art, phage, yeast plasmid,
Plant cell virus, mammalian cell virus or other carriers.In a word, as long as can answer in host's body
Make and stable, any plasmid and carrier can be used.One key character of expression vector is to usually contain
Origin of replication, promoter, marker gene and translation control element.
Method well-known to those having ordinary skill in the art can be used for building the volume of anionic trypsin mutant containing people
Code dna sequence and the expression vector of suitable transcription/translation control signal.These methods include vitro recombination
Dna technology, dna synthetic technology, In vivo recombination technology etc..Described dna sequence can effectively connection
In suitable promoter in expression vector, to instruct mrna to synthesize.Expression vector also includes translating
The ribosome binding site beginning and transcription terminator.
Additionally, expression vector preferably comprises one or more selected markers, it is used for selecting to provide
The phenotypic character of the host cell of conversion, the such as dihydrofolate reductase of eukaryotic culture, neomycin
Resistance and green fluorescent protein (gfp), or resist for colibacillary kanamycin or ampicillin
Property.
Comprise the carrier of above-mentioned suitable dna sequence and suitable promoter or control sequence, Ke Yiyong
In converting suitable host cell, allow it to marking protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryotic cell, such as yeast cells；
Or higher eucaryotic cells, such as plant cell.Representative example has: escherichia coli, streptomyces, agriculture
Bacillus；Fungal cell's such as yeast；Plant cell etc..
When the polynucleotide of the present invention are expressed in higher eucaryotic cells, if inserting enhancer sequence in the carrier
Transcription will be made during row to be strengthened.Enhancer is the cis-acting factors of dna, generally about has 10
To 300 base pairs, act on promoter with the transcription of enhancing gene.
Persons skilled in the art are aware that how to select suitable carrier, promoter, enhancer and host
Cell.
Can be carried out with routine techniquess well known to those skilled in the art with restructuring dna transformed host cell.When
When host is prokaryote such as escherichia coli, the competent cell that can absorb dna can be after exponential phase of growth
Harvest, use cacl2Method is processed, and step used is generally well-known in the art.Another kind of method is to use
mgcl2.If necessary, conversion also can be carried out with the method for electroporation.When host is eukaryote, optional
With following dna transfection method: calcium phosphate precipitation, conventional mechanical methods such as microinjection, electricity is worn
Hole, liposome packaging etc..
The transformant obtaining can be cultivated with conventional method, the albumen of the coded by said gene of the expression present invention.Root
According to host cell used, in culture, culture medium used is selected from various conventional mediums.It is being suitable to place
Cultivated under conditions of chief cell growth.After host cell growth is to suitable cell density, with closing
Suitable method (as temperature transition or chemical induction) induces the promoter of selection, when cell is further cultured for one section
Between.
As another example of the present invention, produce the mutation of people's anionic trypsin by genetic engineering means
Body, such as utilizes any suitable genetic engineering bacterium to produce described people's anionic trypsin mutant,
Separate described people's anionic trypsin mutant.
Recombiant protein in the above methods can be expressed in the cell or on cell membrane or is secreted into thin
Extracellular.If necessary, can be divided by various separation methods using its physics, chemistry and other characteristics
From the albumen with purification of Recombinant.These methods are well-known to those skilled in the art.The example of these methods
Son includes but is not limited to: conventional renaturation process, processed with protein precipitant (salting-out method), centrifugation,
The broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion exchange layer
The combination of analysis, high performance liquid chroma- tography (hplc) and other various liquid chromatography (LC) technology and these methods.
As the optimal way of the present invention, described mutant be containing s121a or r122l or they
Complex mutation body, the complex mutation body containing s139c-s206c and s121a and/or r122l, these
People's anionic trypsin mutant has good stability, and it is ideal to show as temperature stability,
Ph stability is ideal, and biological activity is high.Therefore, this mutant is for widening tryptic reality
Border application has important value.
Hplc analysis is carried out to the mutant of the present invention, obtains the single β-trypsin of peak type, do not contain or
Less α-the trypsin containing degraded.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for
The present invention is described rather than limits the scope of the present invention.The reality of unreceipted actual conditions in the following example
Proved recipe method, generally writes according to normal condition such as j. Pehanorm Brooker etc., Molecular Cloning:A Laboratory guide, the 3rd
Version, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.
Embodiment 1, the expression of mutant s139c-s206c and identification
1st, point mutation design
On the basis of wild type human anionic trypsin sequence, design multiple point mutation, mutant is such as
Under:
(ht2-) s139c mutant: on the basis of seq id no:1 (ht2), the 139th is mutated by s
For c；
(ht2-) s139c-s206c mutant: on the basis of seq id no:1 (ht2), the 139th by s
Sport c, the 206th sports c by s；
(ht2-) s139c-s206c-r122l mutant: on the basis of seq id no:1 (ht2), the 139th
Position sports c by s, and 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 is mutated by s
For a；
(ht2-) r122l mutant: on the basis of seq id no:1 (ht2), the 122nd is mutated by r
For l；
(ht2-) s139c-s206c-s121a mutant: on the basis of seq id no:1 (ht2), the 139th
Position sports c by s, and 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 by s
Sport a, the 122nd sports l by r；
(ht2-) s139c-s206c-s121a-r122l mutant: on the basis of seq id no:1 (ht2),
139th sports c by s, and the 206th sports c by s, and the 121st sports a by s, the
122 sport l by r.
Ht1-c139s-c206s mutant: on the basis of seq id no:2 (ht1), the 139th by c
Sport s.
Sheng Gong Bioisystech Co., Ltd is transferred to prepare the coded sequence of the polypeptide of above-mentioned point mutation (through large intestine bar
The codon optimization of bacterium preference), it is inserted in the ndei/hind iii site of pet-32a plasmid, sequencing is just
After really, can get the recombiant plasmid of point mutation, for recombinant expressed.
2nd, the expression identification of recombiant plasmid
Restructuring matter by the coded sequence of the people's anionic trypsin inserting point mutation of aforementioned preparation
Grain, proceeds in e.coli bl21 (de3) competent cell.Put in 37 DEG C of constant incubators after coated plate,
Put culture 12-16h.Picking single bacterium colony is placed in 30ml shaking flask respectively, treats od600During=0.5-0.6, plus
Enter iptg induction, after 37 DEG C of culture 4h, carry out sds-page identification.Result is shown in Fig. 2.
3rd, ultrasonication
By the bacterium solution after inducing culture, it is centrifuged 20min, collects thalline in 3000rpm, uses 50mmol/l
Thalline is fully suspended by tris-hcl ph 8.0 buffer.The bacterium solution having suspended is placed in Ultrasonic Cell Disruptor,
Work 5s, is spaced 5s, ultrasonic power 200w, circulates 99 times.Will broken after bacterium solution in 12000rpm,
4 DEG C of centrifugation 10min, separate supernatant precipitation.Supernatant precipitation is taken to carry out sds-page identification respectively.
4th, the experimental procedure of western blot
Protein electrophoresises glue is placed on pvdf film, is covered with filter paper, be placed in transfer 1-2 hour in electric turn trough
(1ma-2ma/cm2,10%gel), transfer adds the confining liquid containing 5% skim milk, room temperature after terminating
After incubation 1 hour, add after dilution one to resist, incubated at room 1 hour, wash off one anti-after, according to institute
Plus one anti-add dilution after two anti-incubation 2 hours, by ecl develop the color developing fixing, observed result.
Result is shown in Fig. 3.
5th, result
The plasmid comprising encoding mutant body s139c-s206c is converted to escherichia coli jm109 (de3) and
Carry out expression identification in rosetta (de3) expression bacterium, such as shown in Fig. 2 (a), have no obvious band of expression.
But by induction after and induce before swimming lane compare relatively from the point of view of, have apparent degraded in below 14kda
Band.
By in s139c-s206c gene cloning to pet-32a plasmid, select respectively 15 DEG C, 25 DEG C, 37
Carry out abduction delivering identification, such as Fig. 2 (c) under DEG C temperature conditionss.Find in the precipitation at 15 DEG C and 25 DEG C
Below 14kda has an obvious band of expression, but with destination protein position 29kda at be not inconsistent.37℃
In, there is a deeper band in the precipitation of more than 29kda, but do not obtain substantial amounts of in follow-up test
Precipitation.After carried out western blot identification (Fig. 2 (b)).Result shows, can see in 25 DEG C of precipitations
To a small amount of expression, and there is apparent small molecule band, thus infer it may be possible to s139c-s206c
Extremely unstable during expression, thus can degrade.
Embodiment 2, the expression of mutant s139c, s139c-s206c-r122l
Method according to embodiment 1 is built and expression identification.Result is shown in Fig. 3.
By by induction before and after swimming lane be compared after as can be seen that s139c mutant and
S139c-s206c-r122l mutant in 29kda about have an apparent band of expression, its expression
Amount about accounts for more than the 60% of total protein concentration.
Embodiment 3, the purification of s139c and s139c-s206c-r122l
1st, the washing of inclusion body and renaturation
The precipitation that broken bacterium is obtained is resuspended in containing 0.5%triton x-100 (v:v), 20mmol/l
In the solution of tris-hcl ph 8.0,1mmol/l edta, under room temperature stirring 1h after, in 12000rpm,
4 DEG C of centrifugation 10min remove supernatant, are resuspended in 20mmol/l tris-hcl ph 8.0 solution again after collecting precipitation
Middle repeat the above steps are twice.Finally the inclusion body of washes clean 8m urea solution is dissolved, carry out
Dilution refolding.
2nd, purification pretreatment
Enzymatic activity and activation situation in renaturation solution are detected by enzyme activity determination and sds-page.By renaturation solution
12000rpm centrifugation removes precipitation, and supernatant is dialysed to 1mmol/l hcl with 1:10 (v:v).Dialysis 6h
Change outer liquid once, change 4 times altogether.Renaturation solution after dialysis is carried out next step purification.
3rd, cm-ff ion-exchange chromatography
Using 2 × 15cm chromatographic column, it is previously added the water of 1/10 column volume, closing outlet, by cm-ff
Adsorbent resin aqueous suspension becomes the solution of 50% (v:v), is loaded in chromatographic column using Glass rod drain.Using 2
The water of times column volume uses 0.5m naoh solution slow processes 1-2h after rinsing.Water punching with 5 times of column volumes
It is washed till neutrality, then with 1m nacl slow processes 2 column volume.With 20mmol/l naac-hac ph 5.0
Buffer balances 10 column volumes, and renaturation solution is adjusted to ph 5.0 with 200mmol/l naac, connects ultraviolet
Detector starts loading.20mmol/l naac-hac ph 5.0 buffer is used to balance 2 posts after end of the sample
Volume.Contain the 20mmol/l naac-hac of 0～500mmol/l nacl using 10 times of column volumes during eluting
Ph 5.0 buffer continuous gradient eluting.Purpose peak, sequentially determining are collected according to UV-detector change in value
Often pipe od280 and enzyme activity, and calculate the response rate of albumen.
4th, the detection of people's trypsin vigor
N- benzoyl-l- arginine ethyl ester (n-benzoyl-l-arginine ethyl is used in the present invention
Ester, baee) detect people's trypsin vigor as substrate.Because people's trypsin can specific for hydrolysis essence
The peptide bond of propylhomoserin one of carbon tip, therefore n- benzoyl-l- arginine ethyl ester (baee) can be degraded to n- benzoyl
- l- arginine (benzoyl-l-arginine, ba).Under 253nm wavelength, n- benzoyl-l- arginine second
The absorption value of ester (baee) be far smaller than its degradation product n- benzoyl-l- arginine (benzoyl-l-arginine,
ba).Under certain conditions, with the carrying out of catalytic reaction, product ba gradually increases, the purple of system
Outer absorption value is gradually increased, and finally calculates people's pancreas with the variable quantity △ a253nm of ultraviolet absorption value under 253nm
The activity of protease, the enzyme amount making △ a253nm increase by 0.001 per minute is a baee unit.Enzyme
The scale merit of activity is that 1 usp unit is equal to 3 baee units.When preparing baee substrate, make
Buffer is that 25mmol/l tris-hcl ph7.6 contains 0.1mol/l nacl and 0.01mol/l
cacl2.The working concentration of substrate is 25mmol/l, surveys temperature of living and is 25 DEG C.
5th, result
Result is as shown in Figure 4.Through cm-ff ion-exchange chromatography, obtain more pure albumen,
Find during eluting, mutant s139c meeting next small peak of eluting near 100mmol/l nacl,
Substantially there is no enzyme activity it may be possible to the small peptide degraded.Mutant s139c-s206c-r122l then appearance
Relatively more single, and eluting peak relatively early stage binding ability is slightly weaker than mutant s139c.
Through protein electrophoresises identification, the purity of two kinds of albumen has all reached target, has recorded after purification
The ratio of s139c and s139c-s206c-r122l is lived and is respectively 16018.2u/mg and 10332.1u/mg.
Embodiment 4, mutant s139c-s206c-s121a, s121a-r122l, s121a,
The expression of s139c-s206c-s121a-r122l and purification
Mutant s139c-s206c-s121a, s121a-r122l, s121a, s139c-s206c-s121a-
The recombinant expressed and authentication method of r122l is carried out according to embodiment 1.
Recombinant plasmid transformed is entered in escherichia coli bl21 (de3) expression bacterium, carries out expression identification, knot
Fruit as shown in figure 5, near 29kda, mutant s139c-s206c-s121a, s121a-r122l,
S139c-s206c-s121a-r122l and s121a is all expressed.Cannot obtain in embodiment 1
The s139c-s206c mutant of complete expression, but after adding s121a mutation here, obtain
The expression of s139c-s206c-s121a is it is seen then that s121a serves significance for stable protein expression
Effect.
Purification process is carried out with reference to embodiment 3, and result is shown in Fig. 6.
As shown in Fig. 6 (a), through after purification, four kinds of mutants have all obtained relatively purer albumen.From eluting
From the point of view of on curve, s139c-s206c-s121a-r122l binding ability is slightly strong, such as Fig. 6 (c), Qi Tasi
Plant protein binding capacity all close, s121a such as Fig. 6 (d), there is a foreign protein peak in front.
Mutant s139c-s206c-s121a after purification than live for 11000u/mg,
S139c-s206c-s121a-r122l than work for 13500u/mg, s121a-r122l than work is after purification
It is 10000u/mg that 11500u/mg, s121a ratio after purification is lived.
Embodiment 5, the ph stability of ht2 disulfide bond s139c-s206c series mutants
In the present embodiment, carry out ht2 series mutants s139c and s139c-s206c-r122l with wild
The comparison of the ph stability of type ht1 and ht1 mutant.
Enzyme liquid is respectively placed in ph 3～11 buffer, protein concentration controls in 0.5mg/ml, in 25
Activity is measured, buffer is followed successively by 50mmol/l naac-hac (ph 3～6) after water-bath 2h at DEG C,
Tris-hcl (ph 7～8), gly-naoh (ph 9～11), the equal 25 DEG C of preparations of all buffer.With water-bath
Front enzyme liquid initial activity, as 100%, calculates the remnant enzyme activity under corresponding ph.
Mutant s139c, s139c-s206c-r122l, r122l and ht2, ht1 and
The comparative result of the ph stability of ht1-c139s-c206s is as shown in Figure 7.Consider it respectively in difference
Under ph value, the situation of change of enzyme activity after 25 DEG C of water-bath 2h.Ht1 all shows in the range of ph3-11
Good stability, remnant enzyme activity is all more than 80%.Extremely stable in ph3-4 and 10-11, substantially
Do not interfere with enzyme activity, in ph5 about enzyme activity drop to 80%.By contrast, ht2 is only in ph3
Show certain stability, in the range of ph4-11, remnant enzyme activity is no more than 50%, in ph5
And least stable during ph9, remaining living only has 20%.Mutant ht2-r122l improves to a certain extent
The ph stability of ht2, especially alkali resistance, all carry than ht2 wild type in the range of ph7-11
High 20% remnant enzyme activity.The ph of mutant s139c and ht2 stablizes similar temperament, steady to soda acid
Qualitative slightly improve.Ht1-c139s-c206s this to disulfide bond disappearance mutant and ht1 wild type phase
Ph stability declines substantially ratio, and in the range of ph4 and 6-11, remnant enzyme activity is below 50%.
S139c-s206c-r122l mutant shows good stability, in addition to ph9, other ph
Lower remnant enzyme activity is all more than 50%, and enzyme activity is not lost substantially near ph3-4 and ph11.With
Ht2 wild type is compared ph stability and is greatly improved.
Embodiment 6, the ph stability of people's anionic trypsin s121a series mutants compare
In the present embodiment, carry out ht2 series mutants s121a, s139c-s206c-s121a,
The ph of s139c-s206c-s121a-r122l and s121a-r122l and wild type ht1 and ht2 is stable
The comparison of property.
Enzyme liquid is respectively placed in ph 3～11 buffer, protein concentration controls in 0.5mg/ml, in 25
Activity is measured, buffer is followed successively by 50mmol/l naac-hac (ph 3～6) after water-bath 2h at DEG C,
Tris-hcl (ph 7～8), gly-naoh (ph 9～11), the equal 25 DEG C of preparations of all buffer.With water-bath
Front enzyme liquid initial activity as 100%, the remnant enzyme activity under the corresponding ph of computational item.
The ph stability of s121a series mutants is as shown in figure 8, considered it respectively in different ph values
Under, the situation of change of enzyme activity after 25 DEG C of water-bath 2h.Mutant s139c-s206c-s121a,
S121a-r122l, s121a are compared with ht2 wild type, about the same in each ph stability inferior.
S121a-r122l all improves 10% about remnant enzyme activity in the range of ph6-11.But
Ht2-s139c-s206c-s121a-r122l but significantly improves ph stability, from the model of ph3-11
Enclose interior from the point of view of, in addition to ph9, remnant enzyme activity all more than 80%, all than ht1 near ph5 and ph7
Wild type will height, but in ph9, its remnant enzyme activity but have decreased to less than 50%, this be also ht2 and
The general character of its mutant.
Embodiment 7, the temperature stability of people's trypsin disulfide bond mutant compare
In the present embodiment, carry out s139c and s139c-s206c-r122l and wild type ht1 and ht1 and dash forward
The stability of variant s121a compares.
Take the enzyme liquid of 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 12h in 60 DEG C of water-baths, enzyme activity is measured by sampling every 30min, made with the initial activity of enzyme liquid before water-bath
For 100% calculating remnant enzyme activity at each temperature.
See Fig. 9, considered mutant s139c and s129c-s206c-r122l respectively, in 5mmol/l hcl
In (final concentration of protein 1mg/ml) be placed in the situation of change of enzymatic activity in 12h at 4～60 DEG C.Two kinds of mutants
At 4 DEG C, 15 DEG C, 25 DEG C, 37 DEG C, 40 DEG C, in 12h, vigor is relatively stable, and remnant enzyme activity is all 80%
More than.When 50 DEG C, substantially, in 4h, vigor has declined 50%, 12h to mutant s139c activity decrease
Vigor only has the 20% of initial enzyme activity afterwards, by contrast after s139c-s206c-r122l remnant enzyme activity 12h
For 82%.When 60 DEG C, mutant s139c activity decrease more rapid, after 2h, vigor just drops to
After 20%, 4h, vigor almost exhausts.And after s139c-s206c-r122l places 12h at 60 DEG C,
More than 60% activity still can be retained.
Embodiment 8, the comparison of people's trypsin s121a series mutants temperature stability
Method is with embodiment 7.Result such as Figure 10, be several s121a mutants at different temperatures
Activity change situation in (final concentration of protein 1mg/ml) 12h in 5mmol/l hcl.Below 25 DEG C, 12h
Interior, four kinds of mutant activity situations are basicly stable.When 37 DEG C to 50 DEG C, also can remain with 80% residual
Stay activity.60 DEG C, mutant s139c-s206c-s121a-r122l after 12h, still remain 70% with
On vigor, but these three mutants of s121a, s139c-s206c-s121a, s121a-r122l,
Vigor all have dropped 50% about.So this had both included the mutation of disulfide bond, include position of autotomying again
The mutant s139c-s206c-s121a-r122l of point mutation, stability is fine.
Detection two groups of mutants and wild type is together placed at 60 DEG C, in 1mmol/l hcl, after 12h
Remnant enzyme activity, result is as shown in figure 11, at 60 DEG C, after 12h, ht2 wild type complete deactivation,
The activity decrease of ht2-s139c faster also complete deactivation after 12h.Ht2-r122l mutant final residual
Enzyme activity is 50%.Ht1 remnant enzyme activity be 60%, s121a, s139c-s206c-r122l,
The final enzyme activity of s139c-s206c-s121a mutant is suitable therewith, but activity decrease is slower.But
S139c-s206c-s121a-r122l thermostability is fabulous, and remnant enzyme activity is still more than 70%.
The hplc analysis result of embodiment 9, people's trypsin and its series mutants
Hplc detects people's trypsin, and method is as follows:
Mobile phase
Mobile phase a: containing 0.1%h3po4(85%) aqueous solution.
Mobile phase b: containing 0.1%h3po4(85%) acetonitrile.
Using front using, 0.22 μ l membrane filtration is degerming, ultrasonic degassing.
Chromatographic program: with reference to American Pharmacopeia " enzymes used as ancillary material in 2013
Regulation to recombined human trypsin hplc assay method in pharmaceutical manufacturing ".
Using c18 post (ef-c18 (h) 4.6 × 250mm, 3 μm).
Hplc mobile phase elution program:
Sample size is 1-20 μ l, and in each loading, protein content is no less than 50 μ g.Flow velocity is 1.0ml/min.
Column temperature is 40 DEG C.Detection wavelength is 280nm.
Trypsin using various configuration in the standard analysiss trypsin of hplc trypsin American Pharmacopeia
Enzyme, disengaging time is 30min, wherein different according to trypsin configuration, α-trypsin and β-trypsin
Retention time be 12-17 minute.The hcl being separately added into final concentration of 1mol/l before loading enters to sample
Row acidification.
As can be seen that ht2 appearance time is 13.1min from Figure 12 (d), before β-trypsin, have two
Individual small peak is α-trypsin.As shown in Figure 12 (c), but s139c appearance time is essentially identical with ht2 main
Peak is not single it may be possible to sample is not pure or conformation is not single.R122l appearance time is later to be
15.8min peak type is more single, sees Figure 12 (b).Go out from the visible s139c-s206c-r122l of Figure 12 (a)
Peak time is later, is 16.3min, but its main peak is more single, and there is no α-trypsin.
As Figure 13, found by the hplc testing result of relatively several mutants, with r122l site
Mutant appearance time all can become late.And with the addition of disulfide bond, behind r122l and s121a site, go out
Peak time also can be delayed.But all in all peak type is all more single, the pure β-trypsin of comparison all can be obtained.
Without the mutant in s121a or r122l mutational site, such as s139c-s206c-s121a and
Can there is α-trypsin in s121a, but ht2 wild type becomes apparent from, and also can before α-trypsin position
There are more degraded bands.
The all documents referring in the present invention are all incorporated as reference in this application, just as each literary composition
Offer and be individually recited as with reference to like that.In addition, it is to be understood that reading the above-mentioned teachings of the present invention
Afterwards, those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values are same
Fall within the application appended claims limited range.
Claims (10)
1. the mutant of people's anionic trypsin is it is characterised in that the aminoacid sequence of described mutant
Corresponding to seq id no:1, the 121st sports ala and/or the 139th by ser and is mutated by ser
For cys.
2. mutant as claimed in claim 1 is it is characterised in that the aminoacid sequence of described mutant
Also include: corresponding to seq id no:1,
206th sports cys by ser；Or
122nd sports leu by arg；
And, described mutant is not aminoacid sequence such as seq id no:1 aminoacid sequence and the 139th
Position sports cys, the 206th mutant being sported cys by ser by ser.
3. mutant as claimed in claim 1 is it is characterised in that described mutant is:
As the aminoacid sequence of seq id no:1, but wherein the 139th by ser sport cys,
206 by ser sport cys, the 121st ala the 122nd is sported by ser and is sported by arg
leu；Or
As the aminoacid sequence of seq id no:1, but wherein the 139th by ser sport cys,
206 sport cys and the 122nd by ser and sport leu by arg；Or
As the aminoacid sequence of seq id no:1, but wherein the 139th by ser sport cys,
206 sport cys and the 121st by ser and sport ala by ser；Or
As the aminoacid sequence of seq id no:1, but wherein the 121st sports ala and by ser
122 sport leu by arg.
4. detached polynucleotide are it is characterised in that described polynucleotide encoding claim 1-3 is appointed
Mutant described in one.
5. a kind of carrier is it is characterised in that it contains the polynucleotide described in claim 4.
6. a kind of genetically engineered host cell is it is characterised in that it contains described in claim 5
It is integrated with the polynucleotide described in claim 4 in carrier, or genome.
7. a kind of method of the mutant of people's anionic trypsin producing described in claim 1, it is special
Levy and be, including step:
(1) host cell described in culture claim 6, obtains culture；With
(2) separate the people's anionic trypsin mutant described in claim 1 from culture.
8. the purposes of the arbitrary described people's anionic trypsin mutant of claim 1-3, for digesting
Protein or denatured protein, or it is used for cell cultivation process.
9. a kind of method of the people's anionic trypsin stability improving restructuring is it is characterised in that described
Method includes: the 121st of people's anionic trypsin is sported ala and/or the 122nd by ser
Leu is sported by arg.
10. method as claimed in claim 9 is it is characterised in that also include people's anionic trypsin
The 139th sport cys and/or the 206th by ser cys sported by ser.
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| CN102559645A (en) * | 2010-12-30 | 2012-07-11 | 上海雅心生物技术有限公司 | Human anionic trypsin mutant |
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| CN102559645A (en) * | 2010-12-30 | 2012-07-11 | 上海雅心生物技术有限公司 | Human anionic trypsin mutant |
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| 李强: "重组人源阳离子型胰蛋白酶研究", 《中国优秀硕士学位论文全文数据库 基础科技辑》 * |
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| CN110093336A (en) * | 2018-01-29 | 2019-08-06 | 上海雅心生物技术有限公司 | A kind of trypsase and preparation method thereof that nothing is autotomyed |
| CN110093336B (en) * | 2018-01-29 | 2023-03-24 | 上海雅心生物技术有限公司 | Stable trypsin and preparation method thereof |
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