CN110408635A - A kind of application of the nucleic acid constructs containing Streptavidin element in protein expression, purifying - Google Patents

Abstract

The present invention provides a kind of application of nucleic acid constructs containing Streptavidin element in protein expression, purifying, specifically, the present invention provides a kind of nucleic acid constructs, the nucleic acid constructs has from 5 ' to 3 ' Formulas I structure: Z1-Z2-Z3 (I)；In formula, Z1, Z2, Z3 are respectively the element for being used to constitute the construction；Each "-" independently is key or nucleotide catenation sequence；Z1 is the coded sequence of label protein；Z2 is catenation sequence；Z3 is the coded sequence of nothing or foreign protein, nucleic acid constructs of the invention is applied in protein synthesis system (especially external albumen synthetic system) of the invention, the expression and purifying of foreign protein can be completed, and improves the RFU value of synthesized foreign protein.

Description

A kind of nucleic acid constructs containing Streptavidin element is in protein expression, purifying In application

Technical field

The present invention relates to field of biotechnology, and in particular, to a kind of nucleic acid constructs containing Streptavidin element Application in protein expression, purifying.

Background technique

Protein is the important molecule in cell, almost takes part in the functional execution of cell institute.The sequence and knot of albumen Structure is different, determines the difference (1) of its function.In the cell, albumen can be used as enzyme and be catalyzed various biochemical reactions, Ke Yizuo Coordinate the various activities of organism for signaling molecule, energy, transport molecules can be stored, and make organism in the form of biological support It moves (1).In field of biomedicine, protein antibody as targeted drug, be the diseases such as treating cancer important means (1, 2)。

Yeast expression system is as the exogenous protein expression system risen after one kind, due to having both protokaryon and eukaryotic expression system The advantages of system, just obtains increasingly extensive application in genetic engineering field, using this system can high level expression albumen, and have There is posttranslational modification function, therefore is recognized as a kind of strong tool (3,4) for expressing large-scale protein.

Some Streptavidins (Streptavidin), Avidin are being had been found that into the cell from yeast to the mankind (Avidin) label, and the research of Streptavidin, Avidin label for cellular endogenous and insufficient, are primarily due to The Streptavidin label of endogenous cellular will affect the efficiency of initiation protein translation, and hair is easy in purification process Raw non-specific binding (4,5).Relatively simple different from expressing albumen in virus, different endogenous cellular expression albumen do not have There is the general character in sequence and structure, it is generally difficult to carry out the high-throughput screening (5,6) based on combining biotin.

Other than the understanding that the above people synthesize intracellular protein, protein synthesis can also extracellularly into Row.The outer synthesis system of aleuroplast generally refers in the cracking system of bacterium, fungi, plant cell or zooblast, is added MRNA or the components such as DNA profiling, RNA polymerase and amino acid and ATP complete the rapidly and efficiently translation (7,8) of foreign protein. Currently, the commercialization recombinant protein expression system often tested includes E. coli system (E.coli extract, ECE), rabbit Granulophilocyte (Rabbit reticulocyte lysate, RRL), wheat germ (Wheat germ extract, WGE), insect (Insect cell extract, ICE) and source of people system (7).Compared with traditional In vivo recombination expression system, protein Cell free translation system has the advantages that a variety of in vitro, can such as express effect toxic to cell or contain unnatural amino acid (such as D- amino acid) specific proteins, can direct parallel projects multiple proteins simultaneously using PCR product as template, carry out high The research (7,9) of drug flux screening and proteomics.

However, DNA profiling used in synthesizing in vitro does not have Streptavidin element usually, and to different eggs It is white that be designed different label and purification process time and cost all relatively high (10).And eukaryocyte is used at present Streptavidin element in vitro the expression and purification of initiation protein application it is also fewer.

Therefore, there is an urgent need in the art to develop a kind of to can be used in containing for protein expression and purification in eukaryocyte The nucleic acid constructs is applied in external protein expression system by the nucleic acid constructs of Streptavidin, can be more convenient Albumen is purified, improve purification efficiency, reduce cost.

Summary of the invention

The purpose of the present invention is to provide a kind of can be used in protein expression and purification in eukaryocyte contain chain The nucleic acid constructs is applied in external protein expression system by the nucleic acid constructs of mould Avidin, can be conveniently Albumen is purified, purification efficiency is improved, reduces cost.

First aspect present invention provides a kind of nucleic acid constructs, and the nucleic acid constructs has from 5 ' to 3 ' Formulas I knot Structure:

Z1-Z2-Z3 (I)

In formula,

Z1, Z2, Z3 are respectively the element for being used to constitute the construction；

Each "-" independently is key or nucleotide catenation sequence；

Z1 is the coded sequence of label protein；

Z2 is catenation sequence；

Z5 is the coded sequence of nothing or foreign protein.

In another preferred example, the label protein is the label protein of wild type or optimization.

In another preferred example, the label protein is selected from the group: Streptavidin, MBP, GST, Protein, CBP or A combination thereof.

In another preferred example, the label protein is the Streptavidin of optimization.

In another preferred example, the label protein is marked with Biotin.

In another preferred example, there is the label protein (Kd) to be not more than 10^-6Mol/L, (Kd≤10^-6mol/L)；Or 10^-6Mol/L to 10^-17The binding constant of the protein combination Biotin of coding between mol/L.

In another preferred example, the coded sequence of the label protein is selected from the group；

(a) polypeptide (amino acid sequence of Streptavidin: EAGITGTWYNQLGSTF as shown in SEQ ID NO.:2 is encoded IVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINT QWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS polynucleotides)；

(b) sequence polynucleotides as shown in SEQ ID NO.:1 or 4；

(c) homology >=30% of nucleotide sequence and sequence shown in SEQ ID NO.:1 or 4, preferably, 75% is (preferable Ground >=85%, more preferably >=90% or >=95% or >=98% or >=99%) polynucleotides；

(d) end 5' and/or the end 3' of the polynucleotides shown in SEQ ID NO.:1 or 4 truncate or addition 1-60 (preferable Ground 1-30, more preferably 1-10) nucleotide polynucleotides；

(e) polynucleotides complementary with any polynucleotides of (a)-(d).

In another preferred example, the label protein has sequence or its active fragment shown in SEQ ID NO.:2, or Person have with amino acid sequence shown in SEQ ID NO:2 >=30% homology, (homology preferably, >=85%, more preferably >=90% homology；Homology more preferably >=95%；Most preferably, >=97% homology, such as 98% or more, 99% More than) and there is active polypeptide identical as SEQ ID NO.:2 sequence.

In another preferred example, the catenation sequence is the catenation sequence of codon optimization.

In another preferred example, the catenation sequence have be not easy to be formed secondary structure sequence (such as AT-rich sequence, Without hairpin structure, no G-quadruplex etc.), it is not rich in rare codon.

In another preferred example, the catenation sequence can have restriction enzyme site.

In another preferred example, the catenation sequence is selected from the group；

(i) sequence polynucleotides as shown in SEQ ID NO.:3 or 5；

(ii) homology >=75% of sequence shown in nucleotide sequence and SEQ ID NO.:3 or 5 is (preferably >=85%, more Goodly >=90% or >=95% or >=98% or >=99%) polynucleotides；

(iii) end 5' and/or the end 3' of the polynucleotides shown in SEQ ID NO.:3 or 5 truncate or addition 1-60 (compared with Good ground 1-30, more preferably 1-10) nucleotide polynucleotides；

(iv) polynucleotides complementary with (i)-(iii) any polynucleotides.

In another preferred example, the catenation sequence is as shown in SEQ ID NO.:3 or 5.

In another preferred example, the coded sequence of the foreign protein comes from prokaryotes, eucaryote.

In another preferred example, the coded sequence of the foreign protein comes from animal, plant, pathogen.

In another preferred example, the coded sequence of the foreign protein comes from mammal, preferably Primate, grinding tooth Animal, including people, mouse, rat.

In another preferred example, the coded sequence of the foreign protein is selected from the group: coding fluorescence fibroin or fluorescence Plain enzyme (such as firefly luciferase), green fluorescent protein, yellow fluorescence protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate Dehydrogenase, catalase, actin, the exogenous DNA of the Variable Area of antibody, the DNA of luciferase mutant or its group It closes.

In another preferred example, the foreign protein is selected from the group: (such as firefly is glimmering for fluorescent proteins or luciferase Light element enzyme), green fluorescent protein, yellow fluorescence protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, hydrogen peroxide Enzyme, actin, the Variable Area of antibody, luciferase mutation, alpha-amylase, enterocin A, hepatitis C virus E 2 sugar egg White, insulin precurosor, Interferon α A, interleukin-1 ' beta ', lysozyme element, seralbumin, single-chain antibody section (scFV), first Shape parathyrine transporter, tyrosinase, zytase, or combinations thereof.

In another preferred example, 5 ' end upstreams of the nucleic acid constructs further include promoter.

In another preferred example, the promoter is selected from the group: T7 promoter, T3 promoter, SP6 promoter or its group It closes.

In another preferred example, the nucleic acid constructs further include enhancer element, RBS ribosome binding sequence, Spacer intervening sequence, other for rna transcription, translation correlated series, or combinations thereof.

In another preferred example, the enhancer element includes IRES element, RBS element, non-coding sequence or its group It closes.

In another preferred example, IRES element source one or more cells selected from the group below: prokaryotic cell, eukaryon Cell.

In another preferred example, the eukaryocyte includes higher eucaryotic cells.

In another preferred example, the IRES element includes endogenous IRES element and exogenous IRES element.

In another preferred example, IRES element source one or more cells selected from the group below: people (human), in State's hamster ovary cell (Chinese hamster ovary cell, CHO), insect cell (insect), wheat germ (Wheat Germ cells), rabbit granulophilocyte (Rabbit reticulocyte).

In another preferred example, the IRES element is selected from the group: ScGPR1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1, GAA, Omega, Omega10A, or combinations thereof.

Second aspect of the present invention provides a kind of carrier or carrier combination, and the carrier or carrier combination contain the present invention Nucleic acid constructs described in first aspect.

Third aspect present invention provides a kind of genetically engineered cell, one of the genome of the genetically engineered cell or Multiple integrations have in construction described in first aspect present invention or the genetically engineered cell containing the present invention first Carrier described in aspect or carrier combination.

In another preferred example, the genetically engineered cell includes prokaryotic cell, eukaryocyte.

In another preferred example, the eukaryocyte includes higher eucaryotic cells.

In another preferred example, the genetically engineered cell is selected from the group: human archeocyte (such as Hela cell), Chinese hamster Gonad cell, insect cell, wheat germ cell, rabbit granulophilocyte, yeast cells, or combinations thereof.

In another preferred example, the genetically engineered cell is yeast cells.

In another preferred example, the yeast cells is selected from the group: saccharomyces cerevisiae, Kluyveromyces yeast or its group It closes.

In another preferred example, the Kluyveromyces yeast is selected from the group: Kluyveromyces lactis, Marx's Crewe Tie up yeast, more cloth kluyveromyces, or combinations thereof.

One of fourth aspect present invention provides a kind of kit, and the reagent for including in the kit is selected from the group Or it is a variety of:

(a) construction described in first aspect present invention；

(b) carrier described in second aspect of the present invention or carrier combination；With

(c) genetically engineered cell described in third aspect present invention.

In another preferred example, the kit further includes (d) eukaryon external biological synthetic system (outer albumen of such as eucaryote Synthetic system).

In another preferred example, the eukaryon external biological synthetic system is selected from the group: yeast external biological synthetic system, Chinese hamster ovary cell external biological synthetic system, insect cell external biological synthetic system, Hela cells in vitro biology close Architectonical, or combinations thereof.

In another preferred example, the eukaryon external biological synthetic system includes the outer albumen synthetic system of eucaryote.

In another preferred example, the outer albumen synthetic system of the eucaryote is selected from the group: the outer albumen synthetic system of yeast, The external albumen synthetic system of Chinese hamster ovary cell, the external albumen synthetic system of insect cell, Hela cells in vitro albumen close Architectonical, or combinations thereof.

In another preferred example, the kit further includes (e) yeast external biological synthetic system (outer albumen of such as yeast Synthetic system).

In another preferred example, the yeast external biological synthetic system (the outer albumen synthetic system of such as yeast) is Crewe Tieing up yeast external biological synthetic system (the external albumen synthetic system of such as kluyveromyces), (preferably Kluyveromyces lactis is given birth in vitro Object synthetic system) (the external albumen synthetic system of such as Kluyveromyces lactis).

Fifth aspect present invention provides a kind of construction as described in the first aspect of the invention, second aspect of the present invention institute Kit described in carrier or the carrier combination stated, genetically engineered cell or fourth aspect present invention described in third aspect present invention Purposes, for carrying out high-throughput external albumen synthesis.

Sixth aspect present invention provides a kind of foreign protein synthetic method of external high throughput, comprising steps of

(i) in the presence of eukaryon external biological synthetic system, nucleic acid constructs described in first aspect present invention is provided；

(ii) under the suitable conditions, the eukaryon external biological synthetic system of incubation step (i) T1 for a period of time, to close At the foreign protein.

In another preferred example, the method also includes: (iii) is optionally from the eukaryon external biological synthetic system In, separate or detect the foreign protein.

In another preferred example, the eukaryon external biological synthetic system is yeast external biological synthetic system (such as yeast External albumen synthetic system).

In another preferred example, the yeast external biological synthetic system (the outer albumen synthetic system of such as yeast) is Crewe Tieing up yeast external biological synthetic system (the external albumen synthetic system of such as kluyveromyces), (preferably Kluyveromyces lactis is given birth in vitro Object synthetic system) (the external albumen synthetic system of such as Kluyveromyces lactis).

In another preferred example, the coded sequence of the foreign protein comes from prokaryotes, eucaryote.

In another preferred example, the coded sequence of the foreign protein comes from animal, plant, pathogen.

In another preferred example, the coded sequence of the foreign protein comes from mammal, preferably Primate, grinding tooth Animal, including people, mouse, rat.

In another preferred example, the coded sequence of the foreign protein encodes foreign protein selected from the group below: fluorescein It is albumen or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescence protein, aminoacyl tRNA synthetase, sweet Oily aldehyde -3- phosphate dehydrogenase, catalase, actin, the Variable Area of antibody, luciferase mutant, alpha-amylase, Enterocin A, hepatitis C virus E 2 glycoprotein, insulin precurosor, Interferon α A, interleukin-1 ' beta ', lysozyme element, blood Pure albumen, single-chain antibody section (scFV), transthyretin, tyrosinase, zytase, or combinations thereof.

In another preferred example, the foreign protein is selected from the group: (such as firefly is glimmering for fluorescent proteins or luciferase Light element enzyme), green fluorescent protein, yellow fluorescence protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, hydrogen peroxide Enzyme, actin, the Variable Area of antibody, luciferase mutation, alpha-amylase, enterocin A, hepatitis C virus E 2 sugar egg White, insulin precurosor, Interferon α A, interleukin-1 ' beta ', lysozyme element, seralbumin, single-chain antibody section (scFV), first Shape parathyrine transporter, tyrosinase, zytase, or combinations thereof.

In another preferred example, in the step (ii), reaction temperature is 20-37 DEG C, preferably, 22-35 DEG C.

In another preferred example, in the step (ii), reaction time 1-10h, preferably, 2-8h.

Seventh aspect present invention provides a kind of catenation sequence, and the catenation sequence is selected from the group:

(i) sequence polynucleotides as shown in SEQ ID NO.:3 or 5；

(ii) homology >=75% of sequence shown in nucleotide sequence and SEQ ID NO.:3 or 5 is (preferably >=85%, more Goodly >=90% or >=95% or >=98% or >=99%) polynucleotides；

(iii) end 5' and/or the end 3' of the polynucleotides shown in SEQ ID NO.:3 or 5 truncate or addition 1-60 (compared with Good ground 1-30, more preferably 1-10) nucleotide polynucleotides；

(iv) polynucleotides complementary with (i)-(iii) any polynucleotides.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, In This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 shows the basic bioprocess from DNA to protein.

Fig. 2 shows the starting synthesis in protein synthesis system in vitro of 2 kinds of eukaryocyte Streptavidin sequences The Relative Fluorescence Unit values of enhanced green fluorescence protein (Enhanced green fluorescent protein, eGFP) (Relative Fluorescence Units,RFUs)；STN1 (directly transfers natural Streptavidin chain from gene pool Mould Avidin gene order) and STN2 (under the premise of amino acid sequence is immovable according to complete sequence feature use synonymous code The mode of son replacement has modified number of base) it is attached between sequence and enhanced green fluorescence protein by Linker2, shape At STN-Linker (being named as SAN)；Their Relative Fluorescence Unit values have reached 400 or more, wherein reaction 20 hours Relative Fluorescence Unit values respectively reached 570 and 620, NC (Negative Control) is the reality for being added without nucleic acid constructs A group readings is tested, expressing quantity can carry out purification experiment.

Fig. 3 is shown selects different Linker sequences protein outside yeast to close in STN1 and target protein (eGFP) The enhanced green fluorescence protein (Enhanced green fluorescent protein, eGFP) of synthesis is originated in architectonical Relative Fluorescence Unit values (Relative Fluorescence Units, RFUs), NC is the experiment for being added without nucleic acid constructs Group readings, it can be seen that be significantly increased using the Relative Fluorescence Unit values of improved Linker (Linker2 and Linker3).

Fig. 4 shows that biotin beads is added in reaction solution removes combining target foreign protein, and carries out SDS-PAGE Protein adhesive testing result, stripe size is correct and high-visible as the result is shown.STN1, STN2 refer respectively to STN (STN1, STN2) It is not connected to the experimental group of target protein afterwards, SAN1-eGFP, SAN2-eGFP respectively correspond STN (STN1, STN2) linking objective afterwards The experimental group of albumen (eGFP).

Fig. 5 shows that SAN is applied to the basic principle of protein expression purifying, and SAN is together with desired protein coding sequences quilt After transcription and translation, by being specifically bound with biotin beads, to grab target protein in reaction solution.For separation Target protein and SAN segment can carry out endonuclease reaction by being inserted into restriction enzyme site (such as TEV restriction enzyme site) between two segments.

Specific embodiment

After extensive and in-depth study, it by largely screening and groping, has unexpectedly discovered for the first time a kind of containing available In the novel nucleic acids construction of the Streptavidin-Linker sequence of target protein expression and purification, nucleic acid construct of the invention Object by optional 5`UTR (such as: promoter, translation initiation site, yeast sources IRES enhancer, ScGPR1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1 etc.), can be used for target protein expression and purification The Linker (SAN) of the coded sequence of Streptavidin sequence, connection Streptavidin sequence and foreign protein is (preferably close Numeral optimization linker or the optimization containing restriction enzyme site linker) and foreign protein coded sequence and 3`UTR structure At, nucleic acid constructs of the invention is applied in protein synthesis system (especially external albumen synthetic system) of the invention, Can complete the expression and purifying of foreign protein, and the RFU value of synthesized foreign protein may be up to 600 or more, with without The nucleic acid constructs of SAN sequence is compared, and improves 2 times.On this basis, the present inventor completes the present invention.

Protein synthesis system

Protein synthesis refers to biology according to the messenger RNA (mRNA) obtained from DNA (DNA) transcription On hereditary information synthetic proteins matter process.Protein synthesis, which is also known as, translates (Translation), i.e., mRNA points Base puts in order the sequence of amino acid process being changed into protein or polypeptide chain in son.This is the second of gene expression Step, generates the final stage of gene product protein.Different histocytes has different physiological functions, is because of their tables Up to different genes, the protein with specific function is generated, the composition for participating in Protein synthesis is more than 200 kinds, master Wanting body is collectively constituted by mRNA, tRNA, ribosome and related enzyme and rho factor.

The outer synthesis system of aleuroplast generally refers in the cracking system of bacterium, fungi, plant cell or zooblast, MRNA or the components such as DNA profiling, RNA polymerase and amino acid and ATP are added, complete the rapidly and efficiently translation of foreign protein. Currently, the commercialization recombinant protein expression system often tested includes E. coli system (E.coli extract, ECE), rabbit Granulophilocyte (Rabbit reticulocyte lysate, RRL), wheat germ (Wheat germ extract, WGE), insect (Insect cell extract, ICE) and source of people system.Compared with traditional In vivo recombination expression system, external cell-free conjunction Have the advantages that at system it is a variety of, can such as express it is toxic to cell effect or contain unnatural amino acid (such as D- amino acid) spy Different protein, can directly using PCR product as template simultaneously parallel projects multiple proteins, carry out high-flux medicaments sifting and The research of proteomics.

Wherein, yeast (yeast) has both the advantage for cultivating simple efficient protein matter folding and posttranslational modification.Wherein make Brewer yeast (Saccharomyces cerevisiae) and pichia yeast (Pichia pastoris) are to express complicated eukaryotic protein The model organism of matter and memebrane protein, yeast also can be used as the raw material for preparing external translating system.

Kluyveromyces (Kluyveromyces) are a kind of ascospore yeast, kluyveromyces marxianus therein (Kluyveromyces marxianus) and Kluyveromyces lactis (Kluyveromyces lactis) are industrially to make extensively Yeast.Compared with other yeast, Kluyveromyces lactis is had many advantages, such as superpower secretion capacity, preferably big Scale fermentation characteristic, the rank of food safety and there is the ability modified after protein translation simultaneously etc..

In the present invention, a kind of preferred protein synthesis system is protein synthesis in vitro system, in the present invention, body Outer protein synthesis system is not particularly limited, and a kind of preferred protein synthesis in vitro system is kluyveromyces expression system (more preferably, Kluyveromyces lactis expression system).

In the present invention, the protein synthesis in vitro system includes:

(a) yeast cell extract；

(b) polyethylene glycol；

(c) optional Exogenous Sucrose；With

(d) optional solvent, the solvent are water or aqueous solvent.

In a particularly preferred embodiment, external albumen synthetic system provided by the invention includes: that yeast cells mentions Take object, 4- hydroxyethyl piperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphyosphate (ATP), guanopterin nucleoside triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), ispol, phosphocreatine, two Sulphur threitol (DTT), creatine phosphokinase, RNase inhibitor, fluorescein, luciferin enzyme dna, RNA polymerase.

In the present invention, RNA polymerase is not particularly limited, and can be selected from one or more RNA polymerases, typically RNA polymerase is t7 rna polymerase.

In the present invention, ratio of the cell extract in vitro in albumen synthetic system is not particularly limited, usually Shared system is 20-70% to the cell extract in protein synthetic proteins synthetic system in vitro, preferably, 30-60%, More preferably, 40-50%.

In the present invention, the cell extract is free of complete cell, and typical cell extract includes being used for egg Initiation factor and elongation factors that the ribosomes of white translation, transfer RNA, aminoacyl tRNA synthetase, protein synthesis need and Terminate releasing factor.In addition, also especially may be used containing other albumen in some cytoplasm from cell in cell extract Dissolubility albumen.

In the present invention, protein content contained by the cell extract is 10-100mg/mL, preferably 20-80mg/ mL.The measurement protein content method is Coomassie brilliant blue measuring method.

In the present invention, the preparation method of the cell extract is unrestricted, and a kind of preferred preparation method includes Following steps:

(i) cell is provided；

(ii) carrying out washing treatment is carried out to cell, obtains washed cell；

(iii) broken cell processing is carried out to washed cell, to obtain cell crude extract；

(iv) cell crude extract is separated by solid-liquid separation, obtains liquid portion, as cell extract.

In the present invention, the solid-liquid separation method is not particularly limited, and a kind of preferred mode is centrifugation.

In a preferred embodiment, the centrifugation carries out in the liquid state.

In the present invention, the centrifugal condition is not particularly limited, and a kind of preferred centrifugal condition is 5000-100000g, Preferably, 8000-30000g.

In the present invention, the centrifugation time is not particularly limited, and a kind of preferred centrifugation time is 0.5min-2h, compared with Goodly, 20min-50min.

In the present invention, the temperature of the centrifugation is not particularly limited, it is preferred that and the centrifugation carries out at 1-10 DEG C, Preferably, being carried out at 2-6 DEG C.

In the present invention, the carrying out washing treatment mode is not particularly limited, and a kind of preferred carrying out washing treatment mode is to adopt It is handled with cleaning solution in the case where pH is 7-8 (preferably, 7.4), the cleaning solution is not particularly limited, the typical washing Liquid is selected from the group: 4- hydroxyethyl piperazineethanesulfonic acid potassium, potassium acetate, magnesium acetate, or combinations thereof.

In the present invention, the mode of broken cell processing is not particularly limited, at a kind of preferred broken cell Reason includes that high pressure is broken, freeze thawing (such as liquid nitrogen cryogenics) is broken.

Ribonucleoside triphosphote mixture in the protein synthesis in vitro system is adenosine triphyosphate, guanosint Guanosine triphosphate, cytidine triphosphate and uridine diphosphate guanosine triphosphate.In the present invention, the concentration of various mononucleotides does not have Especially limitation, the concentration of usual every kind of mononucleotide are 0.5-5mM, preferably 1.0-2.0mM.

Ispol in the protein synthesis in vitro system may include natural or non-natural amino acids, it may include D type or L-type amino acid.Representative amino acid includes (but being not limited to) 20 kinds of natural amino acids: glycine, alanine, figured silk fabrics Propylhomoserin, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, day Winter amide, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.The concentration of every kind of amino acid Usually 0.01-0.5mM, preferably 0.02-0.2mM, such as 0.05,0.06,0.07,0.08mM.

In preference, the protein synthesis in vitro system also contains polyethylene glycol or its analog.Polyethylene glycol or The concentration of its analog is not particularly limited, in general, the concentration (w/v) of polyethylene glycol or its analog is 0.1-8%, preferably Ground, 0.5-4%, more preferably, 1-2%, with the total weight of the albumen synthetic system.Representative PEG example includes (but simultaneously It is not limited to): PEG3000, PEG8000, PEG6000 and PEG3350.It should be understood that may also include other various for system of the invention The polyethylene glycol (such as PEG200,400,1500,2000,4000,6000,8000,10000) of molecular weight.

In preference, the protein synthesis in vitro system also contains sucrose.The concentration of sucrose is not particularly limited, and leads to Often, the concentration of sucrose is 0.03-40wt%, preferably, 0.08-10wt%, more preferably, 0.1-5wt%, with albumen synthesis The total weight of system.

A kind of particularly preferred protein synthesis in vitro system also contains following components other than cell extract: The 4- hydroxyethyl piperazineethanesulfonic acid that 22mM, pH are 7.4,30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, 1.5-4mM nucleosides three Phosphate mixture, the ispol of 0.08-0.24mM, 25mM phosphocreatine, 1.7mM dithiothreitol (DTT), 0.27mg/mL phosphorus Creatine phosphate kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, the DNA, 0.027- of 8-20ng/ μ l firefly luciferase 0.054mg/mL t7 rna polymerase.

The coded sequence (exogenous DNA) of foreign protein

As used herein, term " coded sequence of foreign protein " is used interchangeably with " exogenous DNA ", refers both to the use of external source In the DNA molecular for instructing protein to synthesize.In general, the DNA molecular is linear or cricoid.The DNA molecular contains There is the sequence of encoding foreign proteins.

In the present invention, the example of the sequence of the encoding foreign proteins includes (but being not limited to): genome sequence, CDNA sequence.The sequence of the encoding foreign proteins also contains promoter sequence, 5 ' non-translated sequences, 3 ' non-translated sequences.

In the present invention, the selection of the exogenous DNA is not particularly limited, in general, exogenous DNA is selected from the group: coding is glimmering Light fibroin or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescence protein, aminoacyl tRNA synthesis Enzyme, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, the exogenous DNA of the Variable Area of antibody, luciferase are prominent The DNA of variant, or combinations thereof.

Exogenous DNA is also selected from the following group: coding alpha-amylase, enterocin A, hepatitis C virus E 2 glycoprotein, pancreas Island element precursor, Interferon α A, interleukin-1 ' beta ', lysozyme element, seralbumin, single-chain antibody section (scFV), thyroxine Transporter, tyrosinase, zytase exogenous DNA, or combinations thereof.

In a preferred embodiment, the exogenous DNA encodes albumen selected from the group below: green fluorescent protein (enhanced GFP, eGFP), yellow fluorescence protein (YFP), Escherichia coli beta galactosidase (β-galactosidase, LacZ), people's lysine-tRNA synzyme (Lysine-tRNA synthetase), human leucine-tRNA synzyme (Leucine-tRNA synthetase), arabidopsis glyceraldehyde 3 phosphate dehydrogenase (Glyceraldehyde-3-phosphate Dehydrogenase), mouse catalase (Catalase), or combinations thereof.

Nucleic acid constructs

First aspect present invention provides a kind of nucleic acid constructs, and the construction has from 5 ' to 3 ' Formulas I structure:

Z1-Z2-Z3 (I)

In formula,

Z1, Z2, Z3 are respectively the element for being used to constitute the construction；

Each "-" independently is key or nucleotide catenation sequence；

Z1 is the coded sequence of label protein；

Z2 is catenation sequence；

Z5 is the coded sequence of nothing or foreign protein.

In the present invention, the selection of the coded sequence of the foreign protein is not particularly limited, in general, the volume of foreign protein Code sequence is selected from the group: coding fluorescence fibroin or luciferase (such as firefly luciferase), green fluorescent protein, yellow The variable region of fluorescin, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody The exogenous DNA in domain, luciferase mutant DNA, or combinations thereof.

The coded sequence of foreign protein can also encode albumen selected from the group below: alpha-amylase, enterocin A, the third type liver Scorching virus E 2 glycoprotein, insulin precurosor, Interferon α A, interleukin-1 ' beta ', lysozyme element, seralbumin, single-chain antibody Section (scFV), transthyretin, tyrosinase, zytase, or combinations thereof.

In addition, the nucleic acid constructs of the invention can be linear, it is also possible to cricoid.The core of the invention Acid construct object can be single-stranded, be also possible to double-strand.The nucleic acid constructs of the invention can be DNA, be also possible to RNA or DNA/RNA heterozygosis.

In a preferred embodiment, shown in the sequence table 1 of nucleic acid constructs of the invention.

In another preferred example, the construction further includes element selected from the group below or combinations thereof: promoter, termination Son, poly (A) element, transhipment element, gene target element, riddled basins, enhancer, resistant gene, transposase coding Gene.

Multiple choices marker gene can be applied to the present invention, including but not limited to: nutrient defect type mark, resistance mark Note, reporter gene label.The application of selective key plays a role the screening of recombinant cell (recon), so that recipient cell Born of the same parents can significantly be distinguished with unconverted cell.Nutrient defect type mark is the marker gene and recipient cell by being transferred to Mutated gene is complementary, so that recipient cell be made to show wild type growth.Resistance marker, which refers to, is transferred to recipient cell for resistant gene In, the gene being transferred to makes recipient cell show drug resistance under certain drug concentration.As preferred embodiment of the invention, application Resistance marker realizes the convenient screening of recombinant cell.

In the present invention, using nucleic acid constructs of the invention in protein synthesis in vitro system of the invention, and Biotin-binding technology is used after the reaction was completed, the expression and purifying of foreign protein can be completed, specifically, using this hair The relative light unit value of enhanced green fluorescence protein amount synthesized by bright nucleic acid constructs is relatively high.

Carrier, genetically engineered cell

The present invention also provides a kind of carriers or carrier to combine, and the carrier contains nucleic acid constructs of the invention.It is preferred that Ground, the carrier are selected from: bacterial plasmid, bacteriophage, yeast plasmid or zooblast carrier, shuttle vector；The carrier is Transposon vector.The method for being used to prepare recombinant vector is well known to those of ordinary skill in the art.As long as it can be in place Duplication and stabilization in main body, any plasmid and carrier are all can be adopted.

Those of ordinary skill in the art can be used well known method building and contain promoter of the present invention and/or mesh Gene order expression vector.These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..

The present invention also provides a kind of genetically engineered cell, the genetically engineered cell contains the construction or load Body or carrier combination or the genetically engineered cell chromosomal integration have the construction or carrier.In another preference In, the genetically engineered cell further includes being integrated with transposase base on the carrier containing encoding transposase gene or its chromosome Cause.

Preferably, the genetically engineered cell is eukaryocyte.

In another preferred example, the eukaryocyte, including but not limited to: (preferably, kluyveromyces are thin for yeast cells Born of the same parents, more preferable Kluyveromyces lactis cell).

Construction or carrier of the invention, can be used for converting genetically engineered cell appropriate.Genetically engineered cell can be with It is prokaryotic cell, such as Escherichia coli, streptomyces, Agrobacterium: or low eukaryocyte, such as plant cell, yeast cells；Or It is higher eucaryotic cells, such as insect cell, zooblast.Persons skilled in the art are aware that how to select load appropriate Body and genetically engineered cell.With recombinant DNA transformation gene engineering cell can with routine techniques well known to those skilled in the art into Row.When host is prokaryotes (such as Escherichia coli), CaCl can be used₂Method processing, it is also possible to which electroporation carries out.Work as host It is eucaryote, following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods (such as microinjection, electricity can be selected Perforation, liposome packaging etc.).The methods of Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as leaf disk method, children can also be used in conversion plant Embryo conversion method, bud infusion method etc..

External high-throughput protein synthesis methods

The present invention provides a kind of protein synthesis methods of external high throughput, comprising steps of

(i) nucleic acid constructs described in first aspect present invention is provided in the presence of albumen synthetic system in vitro；

(ii) under the suitable conditions, the external albumen synthetic system of incubation step (i) T1 for a period of time, to synthesize institute State foreign protein.

In another preferred example, the method also includes: (iii) optionally from the external albumen synthetic system, point From or the detection foreign protein.

Main advantages of the present invention include:

(1) present invention firstly discovers that, by optional promoter, ribosome bind site, IRES, it is used for target protein expression The coded sequence of STN (STN1, the STN2) sequence of purifying and foreign protein is as nucleic acid constructs, applied to of the invention external In protein synthesis system, it can be used for the expression and purification of foreign protein.

(2) Streptavidin-Linker of the invention (SAN) amino acid sequence specific recognition and can efficiently combine beads.The identification joint efficiency of these amino acid sequences does not exceed only traditional specific amino acid, and can apply Protein synthesis in vitro system in yeast sources (especially Kluyveromyces lactis source).

(3) compared with other cells, Kluyveromyces lactis because its safety and high efficiency can be applied to food and The production of medicine field protein, in addition being such as adapted to high-throughput protein synthesis the advantages of protein synthesis in vitro system Screening, synthesizes toxic protein and the time is short at low cost etc., so the external protein of Kluyveromyces lactis cell origin closes Architectonical can also be widely used in related fields.

(4) SAN amino acid sequence provided by the invention can not only promote expression and the purification effect of target foreign protein, More important is the possibility for different proteins synthesis that can increase Kluyveromyces lactis protein synthesis in vitro system Property.

(5) present invention firstly discloses a kind of by containing the Streptavidin- that can be used for target protein expression and purification The novel nucleic acids construction of Linker Sequence composition, in protein synthesis system (especially external albumen compound body of the invention System) it is middle using nucleic acid constructs of the invention, the expression and purifying of foreign protein, and synthesized foreign protein can be completed RFU value may be up to 600 or more, compared with without SAN sequence, improve 2 times, and contain the nucleic acid structure of linker being not optimised It builds object to compare, improves 3 times.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.

Unless otherwise instructed, then material used in the embodiment of the present invention and reagent are commercial product.

Foreign protein is by taking eGFP as an example.

1 eukaryocyte Streptavidin amino acid sequence of embodiment determines

The determination of 1.1Streptavidin amino acid sequence: relevant nucleic acid sequence and corresponding is found out from gene pool Amino acid sequence, and as corresponding biotin sequence, WtStreptavidin sequence (STN1) is directly transferred from gene pool Natural Streptavidin sequence, OurStreptavidin sequence (STN2) then under the premise of amino acid sequence is immovable according to Complete sequence feature has modified number of base by the way of synonym replacement.OurStreptavidin and WildtypeStreptavidin, corresponding amino acid sequence are identical.The sequence of Linker, which determines, depends on amino acid sequence Similar with native protein as far as possible, the homology of corresponding amino acid sequence is not less than 30%, and nucleic acid sequence does not contain strong second level Structure sequence, hairpin structure, G-quadruplex etc. (G/C content is indefinite).Wherein, TEV restriction enzyme site is contained in Linker3 Sequence obtains pure target protein after can be used for digestion.

Associated nucleic acid sequences in 1. Kluyveromyces lactis of table

The building of protein synthesis in vitro system plasmid of the embodiment 2 containing eukaryocyte Streptavidin nucleic acid sequence

The synthesis of 2.1 full genomes: 2 STN nucleic acid sequences (STN1, STN2) of engineer are together in series, and using complete The method of gene chemical synthesis is synthesized.

The building of 2.2 plasmids: using two pairs of primers for 2 STN nucleic acid sequences (STN1, STN2) of engineer, after Sew and expand corresponding STN segment respectively from the DNA fragmentation of synthesis for the primer of 1f and 1b, suffix is that the primer of 2f and 2b is then used In the PCR amplification to pD2P-eGFP plasmid.In the plasmid finally constructed, 2 STN nucleic acid sequences (STN1, STN2) are inserted into Between the AUG initiation codon and eGFP of pD2P-eGFP plasmid.The title of plasmid is respectively as follows: pD2P-STN2_eGFP and pD2P- STN1_eGFP。

Linker2 sequence is inserted into existing plasmid pD2P-STN_eGFP by the method for PCR, be inserted into STN (STN1, STN2) between nucleic acid sequence and foreign protein coded sequence (eGFP), building contains the plasmid of STN-Linker (SAN) respectively PD2P-SAN1_eGFP and pD2P-SAN2_eGFP, title and sequence number are all listed in Table 2.

Specific building process is as follows:

For the plasmid construction of pD2P-STN_eGFP, PCR amplification is carried out respectively using two pairs of primers, and 10 μ L is respectively taken to expand Volume increase object is mixed；1 μ L Dpn I, 37 DEG C of incubation 6h is added into 20 μ L amplified productions；4 μ L of product adds after DpnI is handled Enter in 50 μ L DH5 α competent cells, place 30min on ice, after 42 DEG C of heat shock 45s, places 3min on ice, 200 μ L LB are added 37 DEG C of shaken cultivation 4h of fluid nutrient medium, are coated on the LB solid medium containing Amp antibiotic and are incubated overnight；Picking 6 After monoclonal expands culture, after carrying out sequencing confirmation correctly, extracts plasmid and save.

2. primer sequence of table

The application in protein synthesis system in vitro of embodiment 3:STN nucleic acid sequence

3.1 methods for utilizing PCR, and use primer pD2P_F:CGCGAAATTAATACGACTCACTATAGG (SEQ ID NO.:16 and pD2P_R:TCCGGATATAGTTCCTCCTTTCAG (SEQ ID NO.:17) will be located at T7 and transcribe in all plasmids Segment and pD2P-eGFP segment between homing sequence and termination sequence comprising SAN sequence are expanded.

And the method for the DNA fragmentation ethanol precipitation that amplification obtains is purified and is enriched with: 1/ being added into PCR product Then the 3M sodium acetate (pH5.2) of 10 volumes adds 2.5-3 times of volume (volume is the volume being added after sodium acetate) 95% ethyl alcohol, is placed in and is incubated for 15min on ice；30min is centrifuged with the speed higher than 14000g under room temperature, discards supernatant； It is cleaned using 70% ethyl alcohol, is then centrifuged 15min again, discard supernatant, and dissolved precipitating with ultrapure water, measurement DNA is dense Degree.

3.2, according to operation instruction, the DNA fragmentation of purifying are added in homemade protein synthesis in vitro system.And it will Above-mentioned reaction system is placed in 22-30 DEG C of environment, stationary incubation about 2-6h.After reaction, it is placed in Envision immediately 2120 multi-function microplate readers (Perkin Elmer), reading detect eGFP signal strength or weakness, relative light unit value (Relative Fluorescence Unit, RFU) it is used as active unit, as shown in Figure 1.

3.3, which are added biotin-beads in reaction solution, removes combining target foreign protein, and carries out SDS-PAGE protein adhesive Detection, as shown in Figure 2.

Experimental result

1. the building of protein synthesis in vitro system plasmid

By repeatedly attempting, successfully 4 external protein synthesis system plasmids finally are constructed, including all 2 contain (plasmid designations are pD2P-SAN1_eGFP, pD2P- to the plasmid and 2 plasmids without eGFP detection albumen that eGFP detects albumen SAN2_eGFP, pD2P-STN1 and pD2P-STN2).

The application in protein synthesis system in vitro of 2.SAN sequence

As shown in Fig. 2, 2 SAN sequences of screening cause enhanced green fluorescence egg in protein synthesis system in vitro The Relative Fluorescence Unit values (Relative Fluorescence Units, RFU) that white hair goes out reach high value (reaction 3 hours RFU value reaches 400 or more afterwards), it is suitable with the RFU value of the nucleic acid constructs containing linker3, than the core containing linker1.5 (RFU of the nucleic acid constructs containing linker1.5 is only the external source of the nucleic acid constructs containing linker2 to the RFU of acid construct object The 60% of the RFU of albumen) 3 times are improved, protein purification experiment can be carried out.

The present invention the result shows that: SAN sequence can be used for detecting protein expression purification effect, and can be applied to external In protein synthesis system, the efficiency of initiation protein synthesis can be up to or over common tag sequence.It increases external Synthetic system carries out the selectivity of protein expression way of purification, greatly enhances the availability of protein synthesis in vitro system.

Also, research of the invention also found, simple, the very high 5`-UTR of efficiency by structure, strong promoter (such as T7 starting Son, T3 promoter, SP6 promoter), different IRES elements (such as KLNCE102) and different STN sequences, the groups such as 3`-UTR It closes, also can get extraordinary protein expression and purification effect, wherein the RFU value of target protein (eGFP) is up to 600 or more, with It is compared without SAN sequence, Relative Fluorescence Unit values (RFU) improve 2 times.

Comparative example

In nucleic acid constructs of the invention, no linker, the RFU of foreign protein is 85.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Bibliography

1.Garcia RA,Riley MR.Applied biochemistry and biotechnology.Humana Press,1981.263-264.

2.Fromm HJ,Hargrove M.Essentials of Biochemistry.2012；

3.Kenny E,Hinchliffe E.Yeast expression system.EP；1993.

4.S,Nordlund P,Weigelt J,Hallberg BM,Bray J,Gileadi O,et al.Protein production and purification.Nat Methods.2008；5(2):135–46.

5.IP,Skerra A.Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site.Protein Sci.2002；11(4):883–93.

6.Voss S,Skerra A.Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification.Protein Eng.1997；10(8):975– 82.

7.Katzen F,Chang G,Kudlicki W.The past,present and future of cell- free protein synthesis.Trends Biotechnol.2005；23(3):150–6.

8.Gan R,Jewett MC.A combined cell-free transcription-translation system from Saccharomyces cerevisiae for rapid and robust protein synthesis.Biotechnol J.2014；9(5):641–51.

9.Lu Y.Cell-free synthetic biology:Engineering in an open world.Synth Syst Biotechnol[Internet].2017；2(1):23–7.Available from:http:// linkinghub.elsevier.com/retrieve/pii/S2405805X1730008X

10.Hyre DE,Trong I Le,Merritt EA,Eccleston JF,Green NM,Stenkamp RE,et al.Cooperative hydrogen bond interactions in the streptavidin–biotin system.Protein Sci.2006；15(3):459–67.

Sequence table

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tatgtcctga caggtcgcta tgattctgcc ccggcaactg atggatctgg aacagcgctg 180

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ggtcagtatg ttggaggcgc cgaagctcgt attaatacac agtggctgct gacaagtggt 300

acaactgaag ccaatgcctg gaaatcaacc ctggttggcc atgacacctt tacaaaagtc 360

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Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp

35 40 45

Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val

50 55 60

Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser

65 70 75 80

Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu

85 90 95

Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val

100 105 110

Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

115 120 125

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ggacagtacg tcggaggtgc cgaggctagg ataaacacgc agtggttgct aacatcaggc 300

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Claims (10)

1. a kind of nucleic acid constructs, which is characterized in that the nucleic acid constructs has from 5 ' to 3 ' Formulas I structure:

Z1-Z2-Z3 (I)

In formula,

Z1, Z2, Z3 are respectively the element for being used to constitute the construction；

Each "-" independently is key or nucleotide catenation sequence；

Z1 is the coded sequence of label protein；

Z2 is catenation sequence；

Z5 is the coded sequence of nothing or foreign protein.

2. nucleic acid constructs as described in claim 1, which is characterized in that the label protein is selected from the group: Streptavidin, MBP, GST, Protein, CBP, or combinations thereof.

3. nucleic acid constructs as described in claim 1, which is characterized in that the catenation sequence is selected from the group；

(i) sequence polynucleotides as shown in SEQ ID NO.:3 or 5；

(ii) homology >=75% of sequence shown in nucleotide sequence and SEQ ID NO.:3 or 5 is (preferably >=85%, more preferably >=90% or >=95% or >=98% or >=99%) polynucleotides；

(iii) end 5' and/or the end 3' of the polynucleotides shown in SEQ ID NO.:3 or 5 truncate or addition 1-60 is a (preferably 1-30, more preferably 1-10) nucleotide polynucleotides；

(iv) polynucleotides complementary with (i)-(iii) any polynucleotides.

4. a kind of carrier or carrier combination, which is characterized in that the carrier or carrier combination contain core described in claim 1 Acid construct object.

5. a kind of genetically engineered cell, which is characterized in that one or more sites of the genome of the genetically engineered cell are whole Conjunction contains carrier as claimed in claim 4 or carrier in construction or the genetically engineered cell described in having the right to require 1 Combination.

6. a kind of kit, which is characterized in that the reagent for including in the kit one of is selected from the group or a variety of:

(a) construction described in claim 1；

(b) carrier or carrier combination as claimed in claim 4；With

(c) genetically engineered cell described in claim 5.

7. described in a kind of construction as described in claim 1, carrier as claimed in claim 4 or carrier combination, claim 5 Genetically engineered cell or claim 6 described in kit purposes, which is characterized in that for carrying out high-throughput external albumen Synthesis.

8. a kind of foreign protein synthetic method of external high throughput, which is characterized in that comprising steps of

(i) in the presence of eukaryon external biological synthetic system, nucleic acid constructs described in claim 1 is provided；

(ii) under the suitable conditions, the eukaryon external biological synthetic system of incubation step (i) T1 for a period of time, to synthesize institute State foreign protein.

9. method according to claim 8, which is characterized in that the method also includes: (iii) is optionally from the eucaryote In outer biology synthetic system, the foreign protein is separated or detected.

10. a kind of catenation sequence, which is characterized in that the catenation sequence is selected from the group:

(i) sequence polynucleotides as shown in SEQ ID NO.:3 or 5；

(ii) homology >=75% of sequence shown in nucleotide sequence and SEQ ID NO.:3 or 5 is (preferably >=85%, more preferably >=90% or >=95% or >=98% or >=99%) polynucleotides；

(iii) end 5' and/or the end 3' of the polynucleotides shown in SEQ ID NO.:3 or 5 truncate or addition 1-60 is a (preferably 1-30, more preferably 1-10) nucleotide polynucleotides；

(iv) polynucleotides complementary with (i)-(iii) any polynucleotides.