CN106801047A - A kind of method that cysteine proteinase is prepared by cell free, protein synthesizing system - Google Patents
A kind of method that cysteine proteinase is prepared by cell free, protein synthesizing system Download PDFInfo
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- CN106801047A CN106801047A CN201611257363.9A CN201611257363A CN106801047A CN 106801047 A CN106801047 A CN 106801047A CN 201611257363 A CN201611257363 A CN 201611257363A CN 106801047 A CN106801047 A CN 106801047A
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- cysteine proteinase
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- synthesizing system
- protein synthesizing
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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)
- C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
The invention discloses a kind of method that cysteine proteinase is prepared by cell free, protein synthesizing system, belong to biological technical field.The method is comprised the following steps：1) synthesis of cysteine proteinase gene and clone；2) construction recombination plasmid pET28a AsNODf32；3) acellular albumen expression system is prepared, and expression is carried out in expression system and form cysteine proteinase precipitation；4) to the resuspended precipitation process of cysteine proteinase.The cysteine proteinase gene for synthesizing express by the present invention in cell free, protein synthesizing system, without preparing mRNA and expression quantity is high, and simplification following purification steps.
Description
Technical field
The present invention relates to albumen preparation field, and in particular to one kind prepares cysteine by cell free, protein synthesizing system
The method of protease.
Background technology
One the most important protease family of cysteine proteinase, the widely various physiology courses of involved in plant, example
Such as, the process such as seed sprouting, the development of seedling, Stress responses, plant tissue differentiation and aging.Plant cysteine proteases master
Belong to papain (C1A, papain-like cydteine proteinases) and peas protein enzyme family (C13
The legumains), and it is the most detailed to the former research.Papain much all contains 10-26 hydrophobic amino acid group
Into the signal peptide for being positioned at endoplasmic reticulum.The domain being made up of a 35-150 amino acid in enzyme precursor, encloses enzyme
Avtive spot, while the domain for protein correct folding and position it is also most important.
Cysteine proteinase take part in regulating senescence of the development of root nodule, the Stress responses of root nodule and root nodule etc., and half
Cysteine proteases have played important function in the aging course of root nodule, and do not have grinding for correlation in root nodule crowtoe of shaping
Study carefully result.The present invention have found one from crowtoe and can regulate and control the protease of Nodule senescence, by increasing capacitance it is possible to increase fixed nitrogen enzyme activity,
Increase root nodule diameter, delay senility, final plant strain growth is luxuriant.The present invention at home and abroad has no report.
The content of the invention
For problems of the prior art, system is synthesized by acellular albumen it is an object of the invention to provide a kind of
Controlling for cysteine proteinase method, acellular albumen synthesis expression system need not prepare mRNA and expression quantity is high.
The present invention is achieved through the following technical solutions：One kind prepares cysteine protein by cell free, protein synthesizing system
The method of enzyme, comprises the following steps：
Step (1)：The synthesis of cysteine proteinase gene and clone：
Compared in Lotus corniculatus var. japonicus website with the cysteine proteinase gene AsNODf32 of Chinese milk vetch, looked for
Homologous clone in crowtoe；The special primer of design expands cysteine proteinase gene from crowtoe genome, its
Gene order such as sequence table SEQ ID NO：Nucleotide sequence shown in 1, double enzyme site to the sequence is introduced with PCR method respectively
Two ends；
Step (2)：The structure of recombinant plasmid：
By the PCR genetic fragment enzymes double zyme cuttings that obtain of amplification, carrier pET28a with same enzymes double zyme cutting,
Purpose fragment is reclaimed, then carrier and purpose fragment is connected with DNA ligase, is converted thin to bacillus coli DH 5 alpha competence
In born of the same parents, screening recon obtains recombinant plasmid pET28a-AsNODf32；
Step (3)：Acellular albumen expression system is prepared, the expression system includes following composition：HEPES-KOH is buffered
Liquid, ATP, GTP, UTP, CTP, cAMP, potassium glutamate, ammonium acetate, magnesium acetate, folinic acid, t7 rna polymerase, amino acid,
PET8000, PEP, Escherichia coli extract；The recombinant plasmid that screening in step (2) is obtained is added without thin
Expressed in born of the same parents' protein expression system.
Step (4)：Cysteine proteinase precipitation process：
Being precipitated cysteine proteinase with the weak detergent solution that mass concentration is 0.5%~1.5% carries out resuspended sinking
Form sediment, add a small amount of reducing agent centrifugation concussion to all dissolvings are precipitated, take supernatant, add dispersant to final concentration of 0.2%~
2%, GSSG stand 12~18h to 1~5mM of final concentration, GSSH to 2~8mM of final concentration；The albumen after standing is taken out, it is slow in TE
Dialysed in fliud flushing, centrifugation obtains supernatant, as final cysteine proteinase solution.
Beneficial effects of the present invention are：The process post-processed using the albumen of acellular high efficient expression, to acellular a large amount of
The albumen precipitation of expression be first denatured after renaturation method, prepared the preferable cysteine proteinase of immunocompetence, gram
Take the low expression amount of common expression system and need addition liposome just active problem；The method need not prepare mRNA and
Expression quantity is high, while simplifying following purification steps.
Brief description of the drawings
Fig. 1 is the electrophoretogram of cysteine proteinase prepared by embodiment 1；
Fig. 2 is the electrophoretogram of cysteine proteinase prepared by embodiment 2.
Specific embodiment
The present invention is described in further detail with implementation method below in conjunction with the accompanying drawings.Following examples are only exemplary
, only it is used to do technical scheme further describe in detail, it will be understood by those within the art that,
In the spirit and scope without departing from technical solution of the present invention, the modification or replacement carried out to technical scheme all should be covered at this
In the claims of invention.
The method that the present invention prepares cysteine proteinase by cell free, protein synthesizing system, comprises the following steps：
Step (1)：The synthesis of cysteine proteinase gene and clone：
Compared in Lotus corniculatus var. japonicus website with the cysteine proteinase gene AsNODf32 of Chinese milk vetch, looked for
Homologous clone in crowtoe；The special primer of design expands cysteine proteinase gene from crowtoe genome, its
Gene order such as sequence table SEQ ID NO：Nucleotide sequence shown in 1, double enzyme site to the sequence is introduced with PCR method respectively
Two ends；
Step (2)：The structure of recombinant plasmid：
By the PCR genetic fragment enzymes double zyme cuttings that obtain of amplification, carrier pET28a with same enzymes double zyme cutting,
Purpose fragment is reclaimed, then carrier and purpose fragment is connected with DNA ligase, is converted thin to bacillus coli DH 5 alpha competence
In born of the same parents, screening recon obtains recombinant plasmid pET28a-AsNODf32；
Step (3)：Acellular albumen expression system is prepared, the expression system includes following composition：HEPES-KOH is buffered
Liquid, ATP, GTP, UTP, CTP, cAMP, potassium glutamate, ammonium acetate, magnesium acetate, folinic acid, t7 rna polymerase, amino acid,
PET8000, PEP, Escherichia coli extract；The recombinant plasmid that screening in step (2) is obtained is added without thin
Expressed in born of the same parents' protein expression system.
Step (4)：Cysteine proteinase precipitation process：
Being precipitated cysteine proteinase with the weak detergent solution that mass concentration is 0.5%~1.5% carries out resuspended sinking
Form sediment, add a small amount of reducing agent centrifugation concussion to all dissolvings are precipitated, take supernatant, add dispersant to final concentration of 0.2%~
2%, GSSG stand 12~18h to 1~5mM of final concentration, GSSH to 2~8mM of final concentration；The albumen after standing is taken out, it is slow in TE
Dialysed in fliud flushing, centrifugation obtains supernatant, as final cysteine proteinase solution.
As further preferably, the Escherichia coli extract is the S30 extracted from Escherichia coli with S30 buffer solutions
Extract；The S30 buffer solutions include following composition：
As further preferably, the weak detergent added in precipitation process is SKL, Triton X2100, octyl Portugal
Glucosides, dexycholate, SDS, CTAB, polysorbas20, NP-40 etc., preferably mass concentration are 0.5% SKL solution.
As further preferably, reducing agent is DTT, DTE, GSH, beta -mercaptoethanol etc., preferably DTT.
Embodiment 1
1. the synthesis of cysteine proteinase gene and clone
Compared in Lotus corniculatus var. japonicus website with the cysteine proteinase gene AsNODf32 of Chinese milk vetch, looked for
Homologous clone in crowtoe；The special primer of design expands cysteine proteinase gene from crowtoe genome, its
Gene order such as sequence table SEQ ID NO：Nucleotide sequence shown in 1, used as pcr template, sense primer is the DNA using synthesis
5’-GATCGGATCCCTGCCGAAAGCGCCG-3 ', anti-sense primer is 5 '-ATGAAGCTTAATATCACGCACATAT-3 ', point
Not Yin Ru BamH I and Hind III restriction enzyme site.PCR reaction conditions:95 DEG C of predegeneration 4min, 94 DEG C are denatured 1min, 52 DEG C
Annealing 1min, 72 DEG C of extension 2min, carry out 30 circulations, 72 DEG C of extension 6min.
2. the structure of recombinant plasmid
The genetic fragment that PCR amplifications are obtained is reclaimed and uses restriction endonuclease BamH I and the double digestions of Hind III, and carrier pET28a is same
Sample BamH I and the double digestions of Hind III, reclaim big fragment.Then carrier and purpose fragment are connected with T4 DNA ligases,
With in its conversion bacillus coli DH 5 alpha competent cell.Using LB ampicillin kanamycins plate screenings recombinant conversion,
Picking transformant is bacterium colony PCR, and the conversion daughter colony extraction recombinant plasmid endonuclease digestion to purposeful band further reflects
Determine, and serve Hai Sheng works Bioisystech Co., Ltd to be sequenced, obtain recombinant plasmid pET28a-AsNODf32.
3. acellular albumen expression system is prepared, and the expression system includes following composition：57mM HEPES-KOH are buffered
Liquid, 1.2mM ATP, 0.85mM GTP, 0.85mM UTP, 0.85mM CTP, 0.64mM cAMP, 200mM potassium glutamate, 80mM
Ammonium acetate, 15mM magnesium acetates, 34 μ g/mL folinic acid, 33 μ g/mL t7 rna polymerases, 500 μM of amino acid, account for gross weight 2%
PET8000,33mM PEP, the Escherichia coli extract for accounting for gross weight 24%；By recombinant plasmid pET28a-
AsNODf32 is added in acellular albumen expression system, takes 300 μ L expression system solution in D-TubeTMIn dialysis tubing, it is placed in
In 50mL centrifuge tubes, in 30 DEG C of overnight incubations, centrifugation separates supernatant precipitation, and the beds of precipitation are cysteine proteinase.Its
In, Escherichia coli extract is the S30 extracts extracted from Escherichia coli with S30 buffer solutions；The S30 buffer solutions are included such as
Lower composition：
5. precipitation process
Precipitation is resuspended with PBS, centrifugation, removes supernatant, repeats the step 2 time.With the resuspended precipitation of 1mL0.5%SKL solution,
Plus DTT to final concentration of 5mM, 30 DEG C, 220r, concussion 1h to precipitation all dissolvings.Centrifugation, takes supernatant.Add 20%PEG
4000 to final concentration of 0.2%, 50mM GSSG to final concentration 1mM, 100mM GSSH to final concentration 2mM.Centrifuge tube places 4 DEG C,
Stand 12h.The albumen after standing is taken out, is dialysed in TE buffer solutions 3 days.TE bufferings fluid exchange 2 times during dialysis.Centrifugation, obtains
Supernatant.As final cysteine proteinase solution.Fig. 1 is the electrophoretogram of cysteine proteinase prepared by embodiment 1.
Embodiment 2
Step 1~4 and step 1~4 of embodiment 1 are identical, and wherein precipitation process is as follows in step 5：
Precipitation is resuspended with PBS, centrifugation, removes supernatant, repeats the step 2 time.With the resuspended precipitation of 1mL1.5%SKL solution,
Plus DTT to final concentration of 5mM, 30 DEG C, 220r, concussion 1h to precipitation all dissolvings.Centrifugation, takes supernatant.Add 20%PEG
4000 to final concentration of 2%, 50mM GSSG to final concentration 5mM, 100mM GSSH to final concentration 8mM.4 DEG C of centrifuge tube placement, it is quiet
Put 12h.The albumen after standing is taken out, is dialysed in TE buffer solutions 3 days.TE bufferings fluid exchange 2 times during dialysis.Centrifugation, obtains
Clearly.As final cysteine proteinase solution.Fig. 2 is the electrophoretogram of cysteine proteinase prepared by embodiment 2.
<110>Wuhan Jin Kairui bioengineering Co., Ltd
<120>A kind of method that cysteine proteinase is prepared by cell free, protein synthesizing system
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1380
<212> DNA
<213>Crowtoe
<400> 1
ccacgtggct cgccggtgat taccaccccg ctggaaaccg tggatgcgct ggtggaagaa 60
gcggcgagag ttatgtgcgc ggtggaaagc tatccgcagc cggaaattag ctggacccgt 120
aacaaaattc tgattaaact gtttgatacc cgttatagca ttcgtgaaaa cggccagctg 180
ctgaccattc tgagcgtgga agatagcgat gatggcattt attgctgcac cgcgaacaac 240
ggcgtgggcg gcgcggtgga aagctgcggc gcgctgcagg tgaaaatgaa accgaaaatt 300
acccgtccgc cgattaacgt gaaaattatt gaaggcctga aagcggtgct gccgtgcacc 360
accatgggca acccgaaacc gagcgtgagc tggattaaag gcgatagccc gctgcgtgaa 420
aacagccgta ttgcggtgct ggaaagcggc agcctgcgta ttcataacgt gcagaaagaa 480
gatgcgggcc agtatcgttg cgtggcgaaa aacagcctgg gcaccgcgta tagcaaagtg 540
gtgaaactgg aagtggaaga agaaagcgaa ccggaacagg ataccaaagt gtttgcgcgt 600
attctgcgtg cgccggaaag ccataacgtg acctttggca gctttgtgac cctgcattgc 660
accgcgaccg gcattccggt gccgaccatt acctggattg aaaacggcaa cgcggtgagc 720
agcggcagca ttcaggaaag cgtgaaagat cgtgtgattg atagccgtct gcagctgttt 780
attaccaaac cgggcctgta tacctgcatt gcgaccaaca aacatggcga aaaatttagc 840
accgcgaaag cggcggcgac cattagcatt gcggaatggc gtgaatattg cctggcggtg 900
aaagaactgt tttgcgcgaa agaatggctg gtgatggaag aaaaaaccca tcgtggcctg 960
tatcgtagcg aaatgcatct gctgagcgtg ccggaatgca gcaaactgcc gagcatgcat 1020
tgggatccga ccgcgtgcgc gcgtctgccg catctggcgt ttccgccgat gaccagcagc 1080
aaaccgagcg tggatattcc gaacctgccg agcagcagca gcagcagctt tagcgtgagc 1140
ccgacctata gcatgaccgt gattattagc attatgagca gctttgcgat ttttgtgctg 1200
ctgaccatta ccaccctgta ttgctgccgt cgtcgtaaac agtggaaaaa caaaaaacgt 1260
gaaagcgcgg cggtgaccct gaccaccctg ccgagcgaac tgctgctgga tcgtctgcat 1320
ccgaacccga tgtatcagcg tatgccgctg ctgctgaacc cgaaactgct gagcctggaa 1380
Claims (5)
1. a kind of method that cysteine proteinase is prepared by cell free, protein synthesizing system, it is characterised in that including following
Step：
Step (1)：The synthesis of cysteine proteinase gene and clone：
Compared in Lotus corniculatus var. japonicus website with the cysteine proteinase gene AsNODf32 of Chinese milk vetch, find hundred
Homologous clone in arteries and veins root；The special primer of design expands cysteine proteinase gene from crowtoe genome, its gene
Sequence such as sequence table SEQ ID NO：Nucleotide sequence shown in 1, double enzyme site to the sequence two is introduced with PCR method respectively
End；
Step (2)：The structure of recombinant plasmid：
The genetic fragment enzymes double zyme cutting that PCR amplifications are obtained, carrier pET28a is reclaimed with same enzymes double zyme cutting
Purpose fragment, then connects carrier and purpose fragment with DNA ligase, is converted to bacillus coli DH 5 alpha competent cell
In, screening recon obtains recombinant plasmid pET28a-AsNODf32；
Step (3)：Acellular albumen expression system is prepared, the expression system includes following composition：HEPES-KOH buffer solutions,
ATP, GTP, UTP, CTP, cAMP, potassium glutamate, ammonium acetate, magnesium acetate, folinic acid, t7 rna polymerase, amino acid,
PET8000, PEP, Escherichia coli extract；The recombinant plasmid that screening in step (2) is obtained is added without thin
Expressed in born of the same parents' protein expression system, formed cysteine proteinase precipitation.
Step (4)：Cysteine proteinase precipitation process：
Being precipitated cysteine proteinase with the weak detergent solution that mass concentration is 0.5%~1.5% carries out resuspended precipitation, plus
Enter a small amount of reducing agent centrifugation concussion to all dissolvings are precipitated, take supernatant, add dispersant to final concentration of 0.2%~2%, GSSG
To 1~5mM of final concentration, GSSH to 2~8mM of final concentration, 12~18h is stood；The albumen after standing is taken out, in TE buffer solutions thoroughly
Analysis, centrifugation obtains supernatant, as final cysteine proteinase solution.
2. the method for skeletal muscle receptor tyrosine kinase being prepared by cell free, protein synthesizing system as claimed in claim 1,
It is characterized in that：The Escherichia coli extract is the S30 extracts extracted from Escherichia coli with S30 buffer solutions；The S30
Buffer solution includes following composition：
3. the method for skeletal muscle receptor tyrosine kinase being prepared by cell free, protein synthesizing system as claimed in claim 1,
It is characterized in that：In the step (4), the weak detergent of addition is SKL, Triton X2100, octylglucoside, deoxidation courage
Hydrochlorate, SDS, CTAB, polysorbas20 or NP-40.
4. the method for skeletal muscle receptor tyrosine kinase being prepared by cell free, protein synthesizing system as claimed in claim 1,
It is characterized in that：In the step (5), the dispersant of addition is PEG 4000.
5. the method for skeletal muscle receptor tyrosine kinase being prepared by cell free, protein synthesizing system as claimed in claim 1,
It is characterized in that：In the step (5), reducing agent is DTT, DTE, GSH or beta -mercaptoethanol.
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Cited By (2)
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| CN111607004A (en) * | 2020-06-08 | 2020-09-01 | 中国科学院合肥物质科学研究院 | Method for selectively protecting enzyme cutting sites in trypsin enzyme cutting process |
| CN112708631A (en) * | 2020-11-24 | 2021-04-27 | 清华大学 | Method for synthesizing protein in cell-free system |
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