CN106636033A - Improved cell-free synthesis system and application thereof - Google Patents
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Abstract
The invention discloses an improved cell-free synthesis system, which comprises improvement of a supply reagent. In addition, the invention further discloses an application of an improved cell-free synthesis system in the synthesis of skeletal muscle receptor tyrosine kinase. The application comprises the following steps: (1) synthesizing and cloning of a skeletal muscle receptor tyrosine kinase gene; (2) building of recombinant plasmids; (3) preparing of an escherichia coli S30 extract; (4) semi-continuous cell-free protein synthesis reaction; and (5) deposition treatment. An improved cell-free synthesis method is adopted, the improved cell-free synthesis system can be used for in vitro synthesis of the skeletal muscle receptor tyrosine kinase and the expression efficiency is improved.
Description
Technical field
The invention belongs to albumen preparing technical field, and in particular to a kind of improved cell free translation system and its application.
Background technology
Natural inner membrane protein matter is present in biomembranous lipid environment, but at present the technology of research inner membrane protein matter is
They are dispersed in the environment of the aqueous solution first, are operated with reference to the method for water soluble protein.In order to effectively study
Inner membrane protein matter, it is necessary premise to be dissolved in water environment.Cause inner membrane protein typically by addition detergent at present
Matter and lipid form soluble compound to complete in water.The dissolving of inner membrane protein matter is a rough process
, often there is protein denaturation and/or aggregation in the process in journey.Loss and inactivation in order to avoid protein, the process
Extreme care is needed during optimization.Therefore course of dissolution is the step of processing the most critical of memebrane protein.
Skeletal muscle receptor tyrosine kinase (MuSK) is a kind of antigen protein being present in mammal body, and skeletal muscle is received
Body EGFR-TK is a kind of transmembrane protein related to acetylcholinergic receptor, in the development of skeletal muscle, skeletal muscle acceptor junket
Histidine kinase be when setting up postsynaptic membrane set up major synaptic skeleton primary requirement, myasthenia gravis (myasthenia
Gravis, MG) it is a kind of autoimmune disease being caused by autoantibody, involving neuromuscular junction (NMJ), muscle
Specificity kinase antibody (MuSKAb) plays critically important effect in the onset and PD in myasthenia gravis. closes at present
Effect in skeletal muscle receptor tyrosine kinase antibody in myasthenia gravis is still one of focus for studying in the world, the research
But there is certain demand to skeletal muscle receptor tyrosine kinase. the albumen is difficult expression in common expression system,
Due to stronger hydrophobicity, almost do not express.
The content of the invention
The invention aims to solve the above problems and provide.
The present invention is achieved through the following technical solutions：
1. the synthesis of skeletal muscle receptor tyrosine kinase gene and clone
The cDNA of secretion type expression people's muscle specific kinases is searched from GeneBank, the amino acid of its 24-495AA is taken
Sequence, on the premise of amino acid sequence is not changed, carries out synthesizing base after codon optimization according to e. coli codon preference
Because of sequence.Sequence is shown in sequence table 1, and double enzyme site is introduced respectively to the sequence two ends with PCR method.
2. the structure of recombinant plasmid
The PCR 1.4kb bands glue reclaims that obtain of amplification simultaneously use enzymes double zyme cutting, and carrier pET28a is equally with same interior
Enzyme cutting double digestion, reclaims big fragment.Then connect carrier and purpose fragment with T4DNA ligases, Escherichia coli are converted with it
In DH5 α competent cells.Screening recon obtains recombinant plasmid pET28a-MuSK.
3. the preparation of Escherichia coli S30 extracts
S30 extracts refer to DNA can be transcribed into mRNA and/or by mRNA using S30 buffer solutions from what Escherichia coli were extracted
It is translated as the prepared product of the vitro reactions mixture of polypeptide.The extract is comprising for the biology needed for acellular albumen synthesis
Learn function tRNA, amino acid and ribosomes etc..S30 extracts are it is known in the art that and being described in for example《In Vitro
Synthesis of Protein in Microbial Systems》Geoffrey Zubay,1973.Can be in accordance with the following methods
Obtain Escherichia coli extract.Numerous culture mediums for being suitable to Escherichia coli Growth, such as 2YTPG culture mediums, Ran Houben may be selected
Art personnel are it should be appreciated that many culture mediums can be adapted to this purpose.With suitable cell buffer suspension liquid suspension large intestine bar
Bacterium cell, uses high-pressure homogeneous crusher machine, centrifugation, removes precipitation, obtains Escherichia coli extract.All steps are grasped on ice
Make.Bacterial strain is with BL21 (DE3).
4. semicontinuous acellular albumen synthetic reaction
The semicontinuous acellular albumen synthetic reaction of standard includes following composition：57mM HEPES-KOH (pH8.2),
1.2mM ATP, 0.85mM GTP, 0.85mM UTP, 0.85mM CTP, 0.64mM cAMP, 200mM potassium glutamates, 80mM acetic acid
Ammonium, 15mM magnesium acetates, 34 μ g/mL folinic acid, 6.7 μ g/mL pET28a-MuSK plasmids, 33 μ g/mL T7RNA polymerizations
Enzyme, 20 kinds of amino acid are each 500 μM, 2%PET8000,33mM PEP, and 24% Escherichia coli extract.
For semi-continuous expression system, 300 μ L standard reactions mixtures are loaded on D-TubeTM Dialyzer Midi
In (Thermo, 3.5kDa), 4.5mL supplies reagent (Feeding buffer) is loaded in 50mL Corning centrifuge tubes.By D-
TubeTMDialyzer Midi are put into 50mL centrifuge tubes.Supply reagent does not have template, without t7 rna polymerase, remaining and mark
Quasi- reactant mixture is consistent.Prepare respective reaction liquid and supply liquid, in adding reaction system, 30 DEG C of overnight incubations.Centrifugation, point
From supernatant precipitation, skeletal muscle receptor tyrosine kinase is memebrane protein, because the hydrophobicity of itself, expression is precipitated memebrane protein.
5. precipitation process
Precipitation is resuspended with PBS, centrifugation, removes supernatant, repeats the step 2 time.It is molten with the weak detergents of 1mL 0.5%-1.5%
The resuspended precipitation of liquid, plus a small amount of reducing agent is to final concentration of 5mM.30 DEG C, 220rpm, concussion 1h to precipitation all dissolvings.Centrifugation, takes
Supernatant.Add dispersant to final concentration of 0.2%-2%, 50mM GSSG to final concentration 1-5mM, 100mM GSSH are to final concentration
2-8mM.Centrifuge tube places 4 DEG C, stands 12h.The albumen after standing is taken out, is dialysed in TE buffer solutions 3 days.TE bufferings during dialysis
Fluid exchange 2 times.Centrifugation, obtains supernatant.As final skeletal muscle receptor tyrosine kinase protein solution.
As further, preferably, the S30 buffer solutions include：
Used as further preferably, the weak detergent added in precipitation process is SKL,
Triton X210, octylglucoside, dexycholate, SDS, CTAB, Tween 20, NP40 etc., preferably
SKL, solution concentration is 0.5%.
Used as further preferred, the 50mM GSSG to final concentration 1mM added in precipitation process, 100mM GSSH are to end
Concentration 2mM.
As further preferred, dispersant is added to be PEG 4000 in precipitation process.
As further, preferably, reducing agent is DTT, DTE, GSH, beta -mercaptoethanol etc., preferably DTT.
The invention has the beneficial effects as follows：
(1) skeletal muscle receptor tyrosine kinase protein is expressed using cell-free expression system, overcomes hydrophobic structure expression
Difficult problem, can prepare the protein product that General Expression system can not prepared.
(2) process post-processed using the albumen of acellular high efficient expression, is entered to the albumen precipitation of acellular great expression
The method of renaturation, has prepared the preferable activated protein of immunocompetence after the denaturation of row elder generation, overcomes the low of common acellular expression
Expression and need to add liposome just activated problem.
Description of the drawings
The electrophoretogram of skeletal muscle receptor tyrosine kinase protein prepared by Fig. 1 embodiments one, it is seen that have one obvious at 60kDa
The size of band, size and albumen is basically identical.
The electrophoretogram of skeletal muscle receptor tyrosine kinase protein prepared by Fig. 2 embodiments two, it is seen that have one obvious at 60kDa
The size of band, size and albumen is basically identical.
Specific embodiment
Below in conjunction with the accompanying drawings the present invention is described in further detail with embodiment.Following examples are only exemplary
, only further describe in detail doing to technical scheme, 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.
Embodiment one
1. the synthesis of skeletal muscle receptor tyrosine kinase gene and clone
The mRNA of secretion type expression people's muscle specific kinases is obtained from gene library, the amino acid of its 24-495AA is taken
Sequence carries out synthetic gene sequence after codon optimization according to e. coli codon preference.Sequence is shown in sequence table 1, with what is synthesized
DNA as pcr template,
Upstream primer is, SEQ ID NO：2
Primer sequence is：5 '-GATCGGATCCCTGCCGAAAGCGCCG-3 ',
Downstream primer is, SEQ ID NO：3
Primer sequence is：5 '-ATG AAGCTTAATATCACGCACATAT-3 ',
The restriction enzyme site of BamH I and Hind III is introduced respectively.PCR reaction conditions:95 DEG C of denaturations 4min, 94 DEG C of denaturation
1min, 52 DEG C of 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 PCR 1.4kb bands glue reclaims that obtain of amplification and with restriction endonuclease BamH I and the double digestions of Hind III, carrier pET28a
Equally with BamH I and the double digestions of Hind III, big fragment is reclaimed.Then carrier and purpose fragment are connected with T4 DNA ligases,
Converted in bacillus coli DH 5 alpha competent cell with it.Using LB ampicillin kanamycins plate screenings recombinant conversion,
Picking transformant is bacterium colony PCR, and the conversion daughter colony extraction recombinant plasmid endonuclease digestion of purposeful band is further reflected
Determine, and serve Hai Sheng works Bioisystech Co., Ltd to be sequenced, obtain recombinant plasmid pET28a-MuSK.
3. the preparation of Escherichia coli extract S30
Extract refer to can by DNA be transcribed into mRNA and/or mRNA is translated as polypeptide vitro reactions mixture preparation
Thing.The extract is comprising for the biological function tRNA needed for acellular albumen synthesis, amino acid and ribosomes etc..S30
Extract is it is known in the art that and being described in for example《In Vitro Synthesis of Protein in
Microbial Systems》Geoffrey Zubay,1973.Escherichia coli extract can in accordance with the following methods be obtained.May be selected
Numerous culture mediums for being suitable to Escherichia coli Growth, such as 2YTPG culture mediums, then it will be understood by a person skilled in the art that many
Culture medium can be adapted to this purpose.With suitable cell buffer suspension liquid suspension Bacillus coli cells, high-pressure homogeneous crusher machine is used, from
The heart, removes precipitation, obtains Escherichia coli extract.All steps are operated on ice.Bacterial strain is with BL21 (DE3).
4. semicontinuous acellular albumen synthetic reaction
The semicontinuous acellular albumen synthetic reaction of standard includes following composition：57mM HEPES-KOH (pH8.2),
1.2mM ATP, 0.85mM each of GTP, UTP and CTP, 0.64mM cAMP, 200mM potassium glutamates, 80mM ammonium acetates,
15mM magnesium acetates, 34 μ g/mL folinic acid, 6.7 μ g/mL pET28a-MuSK plasmids, 33 μ g/mLT7RNA polymerases,
500 μM of 20 kinds of amino acid are each 500 μM, 2%PET8000,33mM PEP, and 24% Escherichia coli extract.
For semi-continuous expression system, 300 μ L standard reactions mixtures are loaded on D-TubeTM Dialyzer Midi
In (Thermo, 3.5kDa), 4.5mL supplies reagent (Feeding buffer) is loaded in 50mL Corning centrifuge tubes.By D-
TubeTMDialyzer Midi are put into 50mL centrifuge tubes.Supply reagent does not have template, without t7 rna polymerase, remaining and mark
Quasi- reactant mixture is consistent.
Semi-continuous expression system requires 2 reaction environments, and centre is separated by one and half dialysis membranes, and suitable preparation scale is other
Protein production.Reative cell (Reaction Room) RM includes macromolecule substrate, such as enzyme and nucleic acid.Another supply room
(Feeding Room) FM includes low molecule precursor, such as amino acid, energy production material and nucleotides.FM is by fresh premise
Material introduces RM, while the catabolite for having inhibitory action constantly enters FM from RM.Therefore, semi-continuous expression system yield
Significantly improve, the reaction time extends.Prepare respective reaction liquid and supply liquid, in adding reaction system, 30 DEG C of overnight incubations.From
The heart, separates supernatant precipitation, and skeletal muscle receptor tyrosine kinase is memebrane protein, because the hydrophobicity of itself, expression is memebrane protein
Precipitation.
5. precipitation process
Precipitation is resuspended with PBS, centrifugation, removes supernatant, repeats the step 2 time.With the resuspended precipitation of 1mL 0.5%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 skeletal muscle receptor tyrosine kinase protein solution.
Embodiment two
Step 1-4 as step 1-4 of embodiment one,
5. precipitation process
Precipitation is resuspended with PBS, centrifugation, removes supernatant, repeats the step 2 time.With the resuspended precipitation of 1mL 1.5%SKL solution,
Plus DTT to final concentration of 5mM.30 DEG C, 220rpm, 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 skeletal muscle receptor tyrosine kinase protein solution.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
These embodiments can be carried out with various changes, modification, replacement and modification in the case of the principle and objective that depart from the present invention, this
The scope of invention is limited by claim and its equivalent.
<110>Wuhan Jin Kairui bioengineering Co., Ltd
<120>A kind of improved cell free translation system and its application
<130> 2016
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1416
<212> DNA
<213> Homo sapiens
<400> 1
ctgccgaaag cgccggtgat taccaccccg ctggaaaccg tggatgcgct ggtggaagaa 60
gtggcgacct 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
tatccgcgta acaacattga atatgtgcgt gatatt 1416
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence
<400> 2
gatcggatcc ctgccgaaag cgccg 25
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
atgaagctta atatcacgca catat 25
Claims (8)
1. a kind of improved cell free translation system, it is characterised in that the supply reagent in the cell free translation system is not contained
MRNA templates.
2. a kind of method for preparing skeletal muscle receptor tyrosine kinase, it is characterised in that comprise the following steps that：
(1) synthesis of skeletal muscle receptor tyrosine kinase gene and clone：
The cDNA of secretion type expression people's muscle specific kinases is searched from GeneBank, the amino acid sequence of its 24-495AA is taken
Row, on the premise of amino acid sequence is not changed, according to e. coli codon preference synthetic gene after codon optimization are carried out,
Its nucleotide sequence such as SEQ ID NO:Shown in 1, using the DNA of synthesis as pcr template, design upstream primer and downstream primer,
Double enzyme site is introduced to the gene two ends, enters performing PCR amplification；
(2) structure of recombinant plasmid：
Reclaim the PCR 1.4kb bands glue that obtains of amplification and use enzymes double zyme cutting, carrier pET28a is with same enzymes double zyme
Cut, reclaim big fragment；Connect carrier and purpose fragment with T4DNA ligases, bacillus coli DH 5 alpha competence is converted with it thin
In born of the same parents, screening recon obtains recombinant plasmid pET28a-MuSK；
(3) preparation of Escherichia coli S30 extracts：
E. coli bl21 (DE3) is chosen, with cell buffer suspension liquid suspension Bacillus coli cells, high-pressure homogeneous crusher machine is used,
Centrifugation, removes precipitation, obtains Escherichia coli extract, and all steps are operated on ice；
(4) semicontinuous acellular albumen synthetic reaction：
300 μ L standard reactions mixtures are loaded on into D-TubeTMIn Dialyzer Midi (Thermo, 3.5kDa), 4.5mL supplies
Reagent Feeding buffer are loaded in 50mL Corning centrifuge tubes, by D-TubeTMDialyzer Midi be put into 50mL from
In heart pipe, 30 DEG C of overnight incubations, centrifugation separates supernatant precipitation；
(5) precipitation process：
Precipitation is resuspended with PBS, centrifugation, removes supernatant, repeats the step 2 time, with the weak detergent solution weights of 1mL 0.5%-1.5%
Outstanding precipitation, plus a small amount of reducing agent is to final concentration of 5mM；30 DEG C, 220rpm, concussion 1h take to all dissolvings, centrifugation is precipitated
Clearly；Dispersant is added to final concentration of 0.2%-2%, 50mM GSSG to final concentration 1-5mM, 100mM GSSH to final concentration 2-
8mM；Centrifuge tube places 4 DEG C, stands 12h；The albumen after standing is taken out, is dialysed in TE buffer solutions 3 days；TE buffer solutions during dialysis
Change 2 times, centrifugation obtains supernatant, obtains final skeletal muscle receptor tyrosine kinase protein solution.
3. a kind of improved cell free translation system according to claim 1, it is characterised in that the acellular synthesis system
Supply reagent in system does not contain t7 rna polymerase.
4. a kind of method for preparing skeletal muscle receptor tyrosine kinase according to claim 2, it is characterised in that step
(3) in, the buffer solution is, when cumulative volume is 2000mL:
5. a kind of method for preparing skeletal muscle receptor tyrosine kinase according to claim 2, it is characterised in that step
(5) in, the weak detergent be SKL, Triton X2100, octylglucoside, dexycholate, SDS, CTAB, Tween
One kind in 20, NP40, preferably SKL, solution concentration is 0.5%.
6. a kind of method for preparing skeletal muscle receptor tyrosine kinase according to claim 2, it is characterised in that step
(5) in, the GSSG preferably final concentration of 1mM, the GSSH preferably final concentration of 2mM.
7. a kind of method for preparing skeletal muscle receptor tyrosine kinase according to claim 2, it is characterised in that step
(5) in, the dispersant is PEG 4000.
8. a kind of method for preparing skeletal muscle receptor tyrosine kinase according to claim 2, it is characterised in that step
(5) in, the reducing agent be DTT, DTE, GSH, beta -mercaptoethanol, preferably DTT.
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| CN111378708A (en) * | 2018-12-28 | 2020-07-07 | 康码（上海）生物科技有限公司 | Optimized in-vitro cell-free protein synthesis system and application thereof |
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