CN103382579A - Method used for screening polypeptide in vitro - Google Patents
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- CN103382579A CN103382579A CN2013102822557A CN201310282255A CN103382579A CN 103382579 A CN103382579 A CN 103382579A CN 2013102822557 A CN2013102822557 A CN 2013102822557A CN 201310282255 A CN201310282255 A CN 201310282255A CN 103382579 A CN103382579 A CN 103382579A
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Abstract
The invention belongs to the field of chemical biology technology, and discloses a method used for screening polypeptide in vitro. In order to break the limitation of existing polypeptide intracellular screening technology, the method comprises following steps: a random dsDNA library is constructed, and is transcribed to form mRNA; the mRNA and an oligonucleotide chain are subjected to anneal for complementation, and then in vitro expression is performed by taking the mRNA as a template, wherein the end of the oligonucleotide chain is connected with puromycin; when the expression is about to be completed, puromycin is delivered into a ribosome, a newly generated polypeptide chain is captured by puromycin and a covalent structure is formed; a random library is produced by inverse transcription, wherein in the random library, polypeptide and encoding information cDNA of the polypeptide are combined correspondingly; after screening, a primer is designed so as to subject the obtained cDNA to PCR amplification; and then a next circle of screening is performed so as to obtain the target polypeptide and encoding information of the target polypeptide after a plurality of screening cycles. The screening technologies employed in the method are in vitro, library capacity can reach 1013 to 1015, the system is stable, operation is simple, and screening efficiency is high.
Description
Technical field
The invention belongs to the chemicobiology technical field, relate to the in-vitro screening of polypeptide, be specifically related to build a cDNA-polypeptide libraries and with the method for setting up, the specific objective polypeptide carried out in-vitro screening.
Background technology
According to Darwinian Evolution Theory, what the selection of occurring in nature biomacromolecule and evolution were followed is all the principle of " survival of the fittest, the survival of the fittest in natural selection ", but this process often needs to experience up to ten thousand years, the even longer time.How under lab to simulate the evolutionary process of biomacromolecule, produce fast the molecule of given activity, be a dream of scientists always.
The eighties in last century, the G.P.Smith of University of Missouri at Columbia has set up the display technique that can carry out peptide molecule selection and Study on Evolution in test tube first, be phage display (Phage display), its principle is fusion polypeptide after a kind of capsid polypeptide amalgamation and expression of allogenic polypeptide and phage to be illustrated in the surface of phage, and the DNA of this syzygy of encoding is arranged in the genome of phage.Maximum characteristics and the advantage of display technique of bacteriophage organically combine genotype and phenotype exactly, and namely the particular phenotype of phage surface (polypeptide matter) is corresponding with the coded message (DNA) in phage.If obtain certain specific polypeptide, need only insert the stochastic sequence library in phage genome, carry out specificity screening, then the DNA on the polypeptide that filters out is checked order, namely know the gene order of expressing this polypeptide, this section sequence is transformed in engineering bacteria can realizes large-scale production and application.But all relate to cell transfecting because phage display technology is shown in when building the library and screening, the library capacity is subjected to the impact of transfection efficiency to be limited in 10
9～10
10, reduced library screening efficient, in addition, in a single day phage display library builds up, and is difficult to carry out effective external sudden change and restructuring again, and then has limited the diversity of molecular genetic in the library.After display technique of bacteriophage, the scientist of countries in the world has successively set up again plasmid displaying, bacterium and yeast surface display etc.Yet they all depend on gene expression in vivo, and the capacity in the library of building and molecular diversity finally will be subject to the restriction of the many factors such as transformation efficiency, the interior environment of born of the same parents.So seeking the complete external display systems that not affected by the factors such as cell transfecting and expression becomes inevitable.Under this background, the L.C.Mattheakis of U.S. Afflymax institute and the J.W.Szostak of Harvard Medical School have successively proposed ribosomal display and mRNA shows two kinds of screening methods based on external acellular expression system, but these two kinds of methods are all take RNA as template, formed RNA-polypeptide syzygy is difficult to withstand harsh screening requirement, particularly easily be subject to the degraded of RNA enzyme, thereby affect whole screening process.
The DNA display technique that we propose be a kind of can be at the external Novel screen choosing method that carries out, except not being subjected to the cell transfecting effectiveness affects, its formed DNA-polypeptide syzygy is also more stable.Therefore, DNA shows as a kind of emerging polypeptide triage techniques, will be at new drug development, and the aspects such as protein interaction and proteomics demonstrate application space more widely.
Summary of the invention
The objective of the invention is the limitation for prior art, set up and a kind ofly can screen the method for specific objective polypeptide from large capacity cDNA-polypeptide libraries.
Technical scheme of the present invention is as follows:
The DNA library of chemical synthesis coding peptide library includes the sequences such as T7 promotor, enhanser and initiator codon near the 5' end regions, has added the affinity purification label near the 3' end; At the external t7 rna polymerase that utilizes, DNA is transcribed into mRNA, and includes one section energy and the fixed sequence program of 5' end with the primer formation complementary structure of tetracycline at the 3' of mRNA end; When carrying out vivoexpression, primer and the translation altogether in cell-free translation system of mRNA library with tetracycline, the tetracycline in latter stage of translation can enter rrna and catch newly-generated polypeptide chain and form covalent structure, through reverse transcription, obtain the random library of an express polypeptide and the corresponding combination of its coded message cDNA, be the cDNA-polypeptide libraries, realize genotype and phenotypic combination; When carrying out in-vitro screening, first target is fixed on the solid phase carrier such as magnetic bead, then with it, cDNA-polypeptide libraries that contains target polypeptides is carried out specificity and select; After the screening end, the design pair of primers carries out pcr amplification to the cDNA that obtains, and enters the lower whorl screening, through too much repeating query ring, finally obtains target polypeptides and coded message thereof.
The structure in the DNA library in coded polypeptide involved in the present invention storehouse, first to synthesize one with the single-stranded DNA banks of stochastic sequence by chemical process, design again one and hold the downstream primer of fixed sequence program complementation with single-stranded DNA banks 3', through the pcr amplification of two circulations, obtain can coded polypeptide the double-stranded DNA library in storehouse.
T7 promotor involved in the present invention is the integral part of gene, is the DNA sequence dna of RNA polymerase specific recognition and combination, controls to become from genetic transcription that mRNA's is initial.
Enhanser involved in the present invention is upper a bit of can the combination with polypeptide of DNA, the zone of intensifying genes Transcription.
Initiator codon involved in the present invention refers to begin on mRNA to translate into the starting point of polypeptide, is made of 3 bases, is generally AUG.What wherein procaryotic initiator codon AUG translation was corresponding is formylmethionine, and what Eukaryotic initiator codon AUG translation was corresponding is methionine(Met).
Primer with tetracycline involved in the present invention be one section at the 5' end oligonucleotide sequence with tetracycline, the base numerical control is between 30-60, tetracycline is to modify by chemical process the end that is coupled to oligonucleotide afterwards, because the amino-terminal end gene that adenosine in the structure of tetracycline and aminoacyl-tRNA molecule is connected is similar, can enters ribosomal A site and form covalent structure with the polypeptide chain that is extending when expressing.
Cell-free translation system involved in the present invention refers to not have the external polypeptide translation synthesis system of intact cell, usually utilize cell-free extract that needed rrna, transfer ribonucleic acid, enzyme, amino acid, energy supply system and mineral ion etc. are provided, instruct the synthetic of polypeptide with the mRNA that adds in test tube; Cell-free extract commonly used has rabbit reticulocyte lysate and wheat germ extract etc.; What the present invention used is rabbit reticulocyte lysate, first transcribing template and annealing with the primer of tetracycline, forms complementary structure and joins in rabbit reticulocyte lysate afterwards during reaction, and 30 ℃ of reaction 20min can complete the expression of polypeptide; In order to form more DNA and polypeptide fusions, reaction tubes can be placed in 40min on ice after expressing end, add afterwards Repone K, magnesium chloride to make K
+, Mg
2+Ionic concn reaches respectively 500mM and 50mM and places 50min in room temperature.
Genotype involved in the present invention and phenotypic combination refer to corresponding the combining of polypeptide of genetic information and its coding, and in the present invention, genotype and phenotypic combination are enter rrna and catch the effect that newly-generated polypeptide chain forms covalent structure and realize in the latter stage of expressing by tetracycline.
In the present invention, related target fixedly refers to according to different screening purposes, target is fixed on by chemical process on the solid phase carrier such as magnetic bead and includes and can select with the material of target generation affinity interaction estimating, refers to target is fixed on be used on magnetic bead that the cDNA-polypeptide libraries that contains target polypeptides is carried out specificity and select herein; With the cDNA-polypeptide syzygy of target polypeptides by being separated with target specific binding on solid phase carrier, then with elutriant, cDNA-polypeptide syzygy is eluted from solid phase carrier the performing PCR amplification of going forward side by side, thereby realize the enrichment to the target polypeptides encoding gene.
As used herein, following word/term has following meanings, unless otherwise indicated.
" DNA ": thymus nucleic acid.Be a class with the biomacromolecule of genetic information, by 3', the 5'-phosphodiester bond is formed by connecting, and is the carrier of genetic information by 4 kinds of main deoxynucleotides (dAMP, dGMP, dCMP and dTMP).
" cDNA ": complementary DNA (cDNA).Take mRNA as template, under the existence of suitable primer, through ThermoScript II catalysis that obtain with the single stranded deoxyribonucleic acid mRNA complementation.
" random dsDNA library ": refer to include on each base position and have the double stranded DNA combination that different bases consist of.
" RNA ": Yeast Nucleic Acid.By 3', the polymer that the 5'-phosphodiester bond is formed by connecting by ribonucleotide.
" mRNA ": messenger RNA(mRNA).It is a class singlestranded RNA that can instruct protein synthesis that carries genetic information.
" PCR ": polymerase chain reaction.It is a kind of method of the synthetic specific DNA fragment of external enzymatic, by a few step reaction composition one-period such as high-temperature denatured, low-temperature annealing and thermophilic extensions, loop, make target DNA be able to rapid amplification, have high specificity, highly sensitive, easy and simple to handle, the characteristics such as save time.
" tetracycline ": a kind of microbiotic is widely used as the inhibitor of protein synthesis.AMP structural similitude on its structure and aminoacyl-tRNA 3 ' end, peptidy transeferace can impel amino acid to be combined with tetracycline and form peptide acyl tetracycline, come off from rrna, thereby the protein synthesis reaction is interrupted.
" vivoexpression ": expression biologically is the central dogma according to genetic code, with the decoding that puts in order of base in the messenger RNA(mRNA) molecule of maturation, and the process of the specific amino acid sequence of generation correspondence.Vivoexpression refers to the protein expression process of carrying out in cell-free system.
" primer ": one section short single stranded RNA or DNA fragmentation, can be combined on nucleic acid chains complementary with it zone, its function is the starting point as the Nucleotide polymerization, nucleic acid polymerase can begin synthetic new nucleic acid chains by its 3' end.
" in-vitro screening ": refer to that in the extracellular screening by repeatedly obtains having the molecule of sp act from the random library of nucleic acid or polypeptide.
The method disclosed in the present key is: whole screening process is all to carry out external, and the library capacity is very big, has greatly increased potential target polypeptides selective; On the other hand, due to the effect of tetracycline, the cDNA-polypeptide libraries of formation can make effective polypeptide obtain enrichment and enter the lower whorl screening by pcr amplification after screening, finally obtains target polypeptides by multi-turns screen.The method is easy, stable, efficient, can be applicable to find the polypeptide ligand that RNA, small molecules, protein etc. are new and illustrate polypeptide and the interaction mechanism of medicine in cell.The present invention has advantages of and obviously is better than prior art, and its major advantage comprises:
1. the library capacity is large.Existing display technique of bacteriophage, owing to all relating to cell transfecting when building library and screening, the library capacity is subjected to the impact of transfection efficiency to be limited in 10
9～10
10, reduced library screening efficient, in addition, in a single day phage display library builds up, and is difficult to carry out effective external sudden change and restructuring again, and then has limited the diversity of molecular genetic in the library.And the institute that the present invention relates to is all to carry out in cell-free system in steps, and the size in library is not subjected to the impact of transfection efficiency, can reach 10
13～10
15
2. stability is high.Existing display technique of bacteriophage is polypeptide to be illustrated in the surface of phage, the situation that not folding polypeptide is degraded in bacterium easily appears in screening process, and the expressed polypeptide of the present invention is take cDNA as carrier, and stability is high, can satisfy harsh screening conditions.
3. easy and simple to handle.Existing display technique of bacteriophage must transform through bacterium, phage packaging, and some display systems also will pass through the cross-film secretion process, complex operation, and the present invention does not all relate to these operations.
4. screening efficiency is high.Because the present invention has the advantages such as the library capacity is large, principle is simple, easy and simple to handle, just can complete whole polypeptide screening process in several weeks, screening efficiency improves greatly.
Description of drawings
Fig. 1 is the schematic flow sheet of specific embodiment 1 in-vitro screening polypeptide.
Fig. 2 is the schematic flow sheet that specific embodiment 1 tetracycline is modified.
Fig. 3 is the schematic flow sheet of specific embodiment 1 tetracycline and oligonucleotide linked reaction.
Fig. 4 is the expression of results figure of specific embodiment 1.
Fig. 5 is the reverse transcription figure as a result of specific embodiment 1.
Embodiment
Below in conjunction with accompanying drawing, further illustrate the present invention by example.One skilled in the art will understand that these examples only are used for explanation the present invention, limit the scope of the invention and be not used in.
The flow process of in-vitro screening polypeptide is seen Fig. 1.
(1) build the double-stranded DNA library that comprises stochastic sequence.Synthesize single-stranded DNA banks with stochastic sequence by chemical process, add the pcr amplification that isocyatic downstream primer carries out two circulations, the final double-stranded DNA random library that obtains to comprise T7 promotor, enhanser, initiator codon, stochastic sequence, affinity purification label coding sequence.
The single stranded DNA stochastic sequence:
TAATACGACTCACTATAGGAGGACGAAATG(NNN)9CACCACCACCATCATCATCAGC?TGCGTAACTC
Downstream primer: GAG TTA CGC AGC TGA TGA
Reaction system and PCR condition:
The PCR condition is: 95 ℃ of preheating 1min; 95 ℃ of sex change 30s, 45 ℃ of renaturation 45s, 72 ℃ are extended 45s, 2 circulations.
(2) in-vitro transcription.Take double-stranded DNA as template, to transcribe under the polysaccharase effect and obtain mRNA, reaction system is as follows:
(3) vivoexpression of polypeptide.Carry out take mRNA as template first making itself and end with the oligonucleotide chain annealing complementation of tetracycline before the polypeptide translation, add rabbit reticulocyte lysate to express, reaction adds Repone K, magnesium chloride to make K after finishing
+, Mg
2+Ionic concn reaches respectively 500mM and 50mM and places 50min in room temperature.Take a small amount of expression system as example, whole reactions steps and system are as follows:
When carrying out great expression, reaction system is amplified in proportion as required and is got final product.
(4) reverse transcription.After expressing end, directly add ThermoScript II in expression system, the synthetic cDNA take mRNA as template, reaction system is as follows:
(5) in-vitro screening and PCR enrichment.Target is fixed on is used on magnetic bead the cDNA-polypeptide libraries that contains target polypeptides is carried out the specificity selection, with the cDNA-polypeptide syzygy of target polypeptides by being separated with target specific binding on solid phase carrier, then with elutriant, cDNA-polypeptide syzygy is eluted from solid phase carrier the performing PCR amplification of going forward side by side, thereby realize the enrichment to the target polypeptides encoding gene, the PCR system is as follows:
Upstream primer sequence: TAATACGACTCACTATAGGAGGACGAAATG
Downstream primer sequence: GAG TTA CGC AGC TGA TGA
The PCR condition is: 95 ℃ of preheating 1min; 95 ℃ of sex change 30s, 45 ℃ of renaturation 45s, 72 ℃ are extended 45s, 25 circulations.
The chemically modified of embodiment 2, tetracycline
Tetracycline is seen Fig. 2 through the final product that chemically modified obtains.Concrete operation step is as follows: a. is dissolved in CH with the 100mg tetracycline
3CN (2ml), 0 ℃ of stirring adds Et
3N (60ul), the solution clarification is dissolved in CH with Fmoc-oSu (72mg)
3CN (1ml), 0 ℃ drops in reaction solution, continues to stir, and becomes white opacity liquid after 30min, and the reaction times is about 0 ℃ of 2h, RT1h, reaction finishes rear with funnel filtered and recycled white solid product P1.B. 150mg P1 is dissolved in the 2ml pyridine, fully stirs evenly, add the 200ul triethylamine, 0 ℃ of stirring; 400mg DMTrCl is dissolved in the 2ml pyridine, then is injected in reaction solution with pin, 0 ℃ is stirred to room temperature, crosses post and reclaims product P 2.C. 420mg P2 is dissolved in the 6ml pyridine, adds 10mgDMAP, ice-water bath is cooled to 0 ℃, slowly adds 0.6ml (Ac) with pin
2O, 0 ℃ is stirred 3h, crosses post and reclaims product P 3.D. 380mgP3 is dissolved in the 5ml CH that drying is processed
2Cl
2, ice-water bath is cooled to 0 ℃, adds the 0.2ml dichloro acetic acid, and 0 ℃ is stirred to room temperature, and reaction 2h crosses post and reclaims product P 4.E. claim 30mg P4, the N2 strict protection adds the 250ul pyridine, and 35 ℃ are stirred 15min P4 is dissolved fully, be yellow solution after dissolving, 0 ℃ of stirring adds 35ul DIPEA, stirs, add at last the inferior phosphoryl chloride of 20ul, 0 ℃ of reaction 1h, 10 ℃-15 ℃ reaction 4h, developping agent is crossed post by methylene dichloride: ethyl acetate=1:3 through alkaline post and is reclaimed product P 5; The P5 that obtains will be connected with the DNA of particular sequence.
Tetracycline after embodiment 3, modification is connected with the DNA of one section fixed sequence program, and reactions steps and present widely used solid phase phosphoramidite triester method are basic identical, and coupled product obtains by the high performance liquid phase separation and purification.As shown in Figure 3, contain the polyoxyethylene glycol of 12 carbon near the Spacer18 representative of DNA5' end, the rC representative base that the 5'-3' direction is turned round on oligonucleotide chain.
Embodiment 4, Fig. 4 are the results of carrying out take different mRNA as template after expression of polypeptides.Use isotropic substance
32One section of P mark with the DNA of mRNA template complementary sequence, and be connected with coupled product in embodiment 3 under the existence of clamping plate, obtain can be used for catching the band tetracycline primer of polypeptide, then make the expression of carrying out polypeptide after the mRNA template annealing complementation of itself and different lengths, obtain the DNA-polypeptide syzygy of different lengths.Except different in mRNA length and previous reaction system, reactions steps and condition are all identical.The result is analyzed as follows: swimming lane 1 is the expression of carrying out under the condition that there is no the mRNA template, does not occur DNA-polypeptide syzygy in swimming lane; 2 of swimming lanes are the mRNA template expressions of results that includes 7 amino-acid residue coded messages, and in swimming lane, the top band is DNA-polypeptide syzygy; Swimming lane 3 is the mRNA template expressions of results that contain 16 amino acid coded messages.The results show reasonableness of the present invention and reliability.
Claims (8)
1. the method for an in-vitro screening polypeptide is characterized in that: by building a random dsDNA library and being transcribed into mRNA, first make itself and end complementary with the DNA annealing of tetracycline before carrying out In Vitro Translation take mRNA as template; When translation closed to an end, tetracycline entered rrna and catches newly-generated polypeptide chain and forms covalent structure, through reverse transcription, formed the random library of an express polypeptide and the corresponding combination of its coded message cDNA; After screening finishes, the cDNA that obtains is carried out pcr amplification, and enter the lower whorl screening, warp is the repeating query ring too much, finally obtains target polypeptides and coded message thereof.
2. the method for in-vitro screening polypeptide according to claim 1, it is characterized in that: described random dsDNA library is the fixed sequence program that two ends comprise the encoding gene of T7 promotor, ribosome bind site, affinity purification label, and middle open reading frame includes the double-stranded DNA library of some stochastic sequences.
3. the method for in-vitro screening polypeptide according to claim 1, it is characterized in that: described mRNA transcribes acquisition by dsDNA under the effect of t7 rna polymerase, transcribes external and carries out.
4. the method for in-vitro screening polypeptide according to claim 1, it is characterized in that: described tetracycline is a peptide species matter synthetic inhibitor, the amino-terminal end gene that in structure and aminoacyl-tRNA molecule, adenosine is connected is similar, can enter ribosomal A site and form covalent structure with the polypeptide chain that is extending when expressing.
5. the method for the in-vitro screening polypeptide described according to claim 1 is characterized in that: described oligonucleotide chain contains the above sequence of one section 15 base near the part of 3' end can be complementary with the DNA sequence dna that is connected to the mRNA3' end.
6. the method for the in-vitro screening polypeptide described according to claim 1, it is characterized in that: described vivoexpression refers to carry out common translation after mRNA template and the oligonucleotide chain annealing of end with tetracycline in rabbit reticulocyte lysate, obtains to comprise the DNA-polypeptide syzygy of target polypeptides.
7. the method for the in-vitro screening polypeptide described according to claim 1, it is characterized in that: described reverse transcription refer to tetracycline catch polypeptide matter form DNA-polypeptide syzygy after under effect in ThermoScript II take mRNA as template, be the synthetic cDNA of primer with the oligonucleotide of tetracycline.
8. the method for the in-vitro screening polypeptide described according to claim 1 is characterized in that: described screening refers to the cDNA-polypeptide libraries that contains target polypeptides is carried out specificity select with being fixed in target on the Myoglobin carrier.
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| CN104774923A (en) * | 2015-03-11 | 2015-07-15 | 华中农业大学 | Method for determining transcriptional control complex |
| CN110637086A (en) * | 2017-03-17 | 2019-12-31 | 珂璧斯塔斯株式会社 | Method for producing complex of RNA molecule and peptide, and use thereof |
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| US20080051299A1 (en) * | 1998-12-02 | 2008-02-28 | Peter Lohse | DNA-protein fusions and uses thereof |
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| CN104774923A (en) * | 2015-03-11 | 2015-07-15 | 华中农业大学 | Method for determining transcriptional control complex |
| CN104774923B (en) * | 2015-03-11 | 2017-10-27 | 华中农业大学 | Method for determining transcription regulation complex |
| CN110637086A (en) * | 2017-03-17 | 2019-12-31 | 珂璧斯塔斯株式会社 | Method for producing complex of RNA molecule and peptide, and use thereof |
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