CN110938649A

CN110938649A - Protein synthesis system for improving expression quantity of foreign protein and application method thereof

Info

Publication number: CN110938649A
Application number: CN201811113130.0A
Authority: CN
Inventors: 郭敏; 丁晓辉; 刘显成; 杨宁; 王绍杰; 董颖颖; 王静; 代田纯; 于雪
Original assignee: Kang Code (shanghai) Biological Technology Co Ltd
Current assignee: Kang Code (shanghai) Biological Technology Co Ltd
Priority date: 2018-09-25
Filing date: 2018-09-25
Publication date: 2020-03-31

Abstract

The invention provides a protein synthesis system for improving the expression quantity of exogenous protein and an application method thereof, and particularly relates to a polynucleotide sequence or a vector constructed by inserting a strong promoter sequence in front of a nucleotide sequence for coding eIF4E binding protein, so that the overexpression of eIF4E binding proteins such as Eap1p and p20 is realized, and the exogenous protein synthesis is carried out by using cell lysate containing the modification. The invention provides a protein synthesis system for improving the expression quantity of foreign proteins, and the synthesis system can obviously improve the efficiency of protein translation.

Description

Protein synthesis system for improving expression quantity of foreign protein and application method thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a protein synthesis system for improving the expression level of foreign proteins and an application method thereof.

Background

Proteins are important molecules in cells, and are involved in performing almost all functions of cells. The difference in the sequence and structure of the protein determines the difference in its function. In cells, proteins can catalyze various biochemical reactions as enzymes, can coordinate various activities of organisms as signaling molecules, can support biological morphology, store energy, transport molecules, and move organisms. In cells, regulation of protein translation plays an important role in many processes such as coping with external stress such as nutrient deficiency, and cell development and differentiation. The four processes of protein translation include translation initiation, translation elongation, translation termination and ribosome recirculation, with the most regulated and rate-limiting step being translation initiation [1 ]. Translation initiation by eukaryotic cells can be divided into two broad categories: the traditional approach that "cap structure" relies on and the approach that "cap structure" does not.

During translation initiation in bacteria, the 30S small subunit forms an initiation complex directly adjacent to AUG of the initiation codon, mediated by 3 initiation factors. Eukaryotic translation initiation is complex, requires 11 initiation factors, and ribosomes bind mRNA at different sites than prokaryotic organisms at the initiation of protein synthesis [2 ]. Translation initiation of eukaryotic mRNA having a "cap structure" (m 7GpppN) is mainly carried out by a scanning mechanism depending on the "cap structure" [1-3 ]. Directly interacting with the cap is eIF4F, a translation factor initiation complex consisting of eIF4G protein simultaneously binding RNA helicase eIF4A and eIF 4E. The eIF4E is a cap-binding protein and is a key regulation target point in a eukaryotic protein synthesis pathway. In mammalian cells, proteins 4E-BPs (eIF4E-binding proteins) that interact with eIF4E inhibit translation of cap-dependent mRNA by binding to eIF4E, rendering it unable to bind to eIF4G, thereby preventing the formation of the translation initiation complex eIF4F [4 ]. The human 4E-BP1 protein is one of the substrates of mammalian target of rapamycin (mTOR), and the phosphorylation level is regulated by the mTOR signaling pathway. When the Thr37, Thr46 and Ser65, Thr70 residues are successively phosphorylated by mTORC1, 4E-BP1 will dissociate from eIF4E, thereby promoting the formation of the eIF4F initiation complex and cap-dependent translation initiation [5-6 ]. The mTOR signaling pathway affects translation initiation and protein synthesis in eukaryotic cells and plays an important role in the growth and proliferation of cells.

mTOR is a class of serine/threonine protein kinases closely related to cell proliferation, is evolutionarily well conserved, widely available from yeast to mammals, and responds to a variety of stimuli including growth factors, insulin, nutrients, amino acids, glucose, etc. outside cells [7 ]. The homology between the human TOR protein-encoding gene and yeast is as high as 40% -60% [8 ]. There are no structurally similar proteins in yeast to mammalian 4E-BPs, and functionally similar proteins that bind eIF4E are p20 and EAP1 p. These two proteins have a highly similar 13-residue sequence comprising the binding domain YxxxxL Φ of eIF4E, x being any amino acid residue and Φ being a hydrophobic residue. In addition to this sequence, there is no other similarity between these two proteins, and it is likely that EAP1p has additional functions that further affect the downstream of the TOR signaling pathway [9 ]. For other eukaryotic eIF4E binding proteins, human 4E-BP1, 4E-BP2 and mouse PHAS-I protein sequences have been reported [10 ].

The prepared yeast lysate contains a large amount of protein synthesis and translation machines, and target proteins can be synthesized by adding templates from an external source. However, the original mRNA of yeast will continue to use resources to synthesize non-target hybrid protein, which affects the synthesis efficiency and yield of target protein. According to the invention, through overexpression of Eap1p, eIF4E is competitively combined with eIF4G, the formation of a translation initiation complex eIF4F is reduced, the original translation process of mRNA cap dependence in yeast lysate is inhibited in vitro, and more resources (such as other translation factors, ribosome, amino acid and the like) are used for synthesizing an exogenously added in-vitro protein synthesis system template independent of a cap structure, so that the efficiency and yield of in-vitro protein synthesis are obviously improved.

However, the efficiency of protein translation synthesis in the in vitro biosynthesis system is still low, and thus it is difficult to satisfy the requirement, and there is a need in the art to develop a new system and method for improving the efficiency of foreign protein translation synthesis.

Disclosure of Invention

The invention aims to provide a novel synthesis system and a method capable of improving the translation synthesis efficiency of a foreign protein. Meanwhile, the modified genetically engineered strain and the application thereof are also provided, such as cell lysate (cell extract) of the genetically engineered strain and the application thereof in cell-free protein synthesis.

In a first aspect, the present invention provides a genetically engineered strain for in vitro cell-free protein synthesis, wherein the genome of said genetically engineered strain incorporates a first exogenous gene expression cassette expressing a first nucleic acid construct having a structure of formula I from 5 'to 3':

A1-A2 (I)；

in the formula (I), the compound is shown in the specification,

a1 and A2 are elements for constituting the construct, respectively;

each "-" is independently a bond or a nucleotide linking sequence;

a1 is a promoter element;

a2 is the coding sequence of eIF4E binding protein;

and, expression or activity of eIF4E binding protein is significantly enhanced in the genetically engineered strain.

In another preferred embodiment, the eIF4E binding protein is selected from the group consisting of: EAP1p, p20, eIF4G1, eIF4G2, 4E-BP1, 4E-BP2, PHAS-I or their combination.

In another preferred embodiment, the eIF4E binding protein is derived from yeast.

In another preferred embodiment, the yeast is selected from the group consisting of: saccharomyces cerevisiae, Kluyveromyces yeast, or a combination thereof.

In another preferred embodiment, the yeast of the genus kluyveromyces is selected from the group consisting of: kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polybranhanskii (Kluyveromyces dobzhanskii), or a combination thereof.

In another preferred embodiment, the eIF4E binding protein is selected from the group consisting of: EAP1p, p20, one or a combination thereof.

In another preferred embodiment, the eIF4E binding protein is derived from human or murine.

In another preferred embodiment, the eIF4E binding protein is selected from the group consisting of: 4E-BP1, 4E-BP2, PHAS-I, or a combination thereof.

In another preferred embodiment, the promoter element is selected from the group consisting of: GAPDH1, HXK1, PGK1, TEF1, TIF1, ADH1, SED1 or their combination.

In another preferred embodiment, the promoter element is selected from the group consisting of: one or a combination of pScGAPDH1, pKLHXK1, pKLPGK1, pScPGPK 1, pScSED1, pScADH1 and pKLGAPDH 1.

In another preferred example, the expression or activity of the eIF4E binding protein is significantly enhanced, which means that the expression or activity of the eIF4E binding protein is increased by more than or equal to 30%, preferably, more than or equal to 50%, more preferably, more than or equal to 70%, more preferably, more than or equal to 90%.

In another preferred example, the expression or activity of the eIF4E binding protein is significantly enhanced, which means that the expression or activity of eIF4E binding protein in the engineered strain (E1)/the expression or activity of housekeeping protein or reference protein in the wild-type strain (E2) ≥ 1, preferably ≥ 2, more preferably ≥ 3, as compared to the wild-type strain.

In another preferred embodiment, the significant enhancement of expression or activity of eIF4E binding protein in said strain is achieved by a means selected from the group consisting of: the Crispr technique.

In another preferred embodiment, the element a2 includes coding sequences for wild-type and mutant eIF4E binding proteins.

In another preferred embodiment, said element A2 has the sequence shown in SEQ ID No. 1 or an active fragment thereof or a nucleotide which has more than or equal to 85% homology (preferably more than or equal to 90% homology; etc. preferably more than or equal to 95% homology; most preferably more than or equal to 97% homology, such as more than 98% or more than 99% homology) with the nucleotide sequence shown in SEQ ID No. 1 and which has the same activity as the sequence shown in SEQ ID No. 1.

In another preferred embodiment, the eIF4E binding protein is an eIF4E binding protein from a cell (e.g., yeast).

In another preferred embodiment, the eIF4E binding protein is an eIF4E binding protein from a cell (e.g., human or murine).

In another preferred embodiment, the cell is selected from the group consisting of: mammalian cells (e.g., HF9, Hela, CHO, HEK293), plant cells, yeast cells, insect cells, or combinations thereof.

In another preferred embodiment, the cell is selected from the group consisting of: hela, CHO, HF9, E μ Myc, HEK293, BY-2, yeast, wheat germ cells, rabbit reticulocytes, or combinations thereof.

In another preferred embodiment, the element a2 is a coding sequence for eIF4E binding protein derived from a cell (e.g., yeast).

In another preferred example, the a1 and a2 are derived from cells (such as yeast).

In another preferred embodiment, the eIF4E binding protein is derived from yeast from one or more sources selected from the group consisting of: pichia pastoris, Kluyveromyces, preferably from Kluyveromyces.

In another preferred example, the kluyveromyces includes kluyveromyces marxianus, and/or kluyveromyces lactis.

In another preferred embodiment, the nucleotide sequence encoding the eIF4E binding protein is as shown in SEQ ID No. 1.

In another preferred example, the protein sequence of the eIF4E binding protein has the sequence shown in SEQ ID NO. 2 or an active fragment thereof, or a polypeptide which has more than or equal to 85% homology (preferably more than or equal to 90% homology; etc. preferably more than or equal to 95% homology; most preferably more than or equal to 97% homology, such as more than 98% or more than 99%) with the polypeptide shown in SEQ ID NO. 2 and has the same activity with the sequence shown in SEQ ID NO. 2.

In another preferred example, the protein sequence of the eIF4E binding protein is shown in SEQ ID No. 2.

In another preferred embodiment, the strain is selected from the group consisting of: a kluyveromyces strain, a pichia strain, a saccharomyces cerevisiae strain, a schizosaccharomyces strain, or a combination thereof.

In a second aspect, the invention provides a use of the genetically engineered strain of the first aspect of the invention to improve the efficiency of in vitro protein synthesis.

In a third aspect, the present invention provides a cell-free cell extract derived from the genetically engineered strain of the first aspect of the present invention.

In another preferred embodiment, the cell extract is a soluble cell extract.

In another preferred embodiment, the cell extract is derived from one or more cells selected from the group consisting of: mammalian cells (e.g., HF9, Hela, CHO, HEK293), yeast cells, plant cells, insect cells, or combinations thereof.

In another preferred embodiment, the cell extract is derived from one or more cells selected from the group consisting of: hela, CHO, HF9, E μ Myc, HEK293, BY-2, yeast, wheat germ cells, rabbit reticulocytes, or combinations thereof.

In another preferred embodiment, the cell extract comprises a yeast cell extract.

In another preferred embodiment, the yeast cell is a yeast from one or more sources selected from the group consisting of: pichia pastoris, Kluyveromyces, or combinations thereof; preferably, the yeast cell comprises: kluyveromyces, more preferably Kluyveromyces marxianus and/or Kluyveromyces lactis.

In another preferred embodiment, the yeast cell extract is an aqueous extract of yeast cells.

In another preferred embodiment, the yeast cell extract does not contain long-chain nucleic acid molecules endogenous to yeast.

In another preferred embodiment, the yeast cell extract is prepared by a method comprising the steps of:

(i) providing a yeast cell;

(ii) washing the yeast cells to obtain washed yeast cells;

(iii) performing cell breaking treatment on the washed yeast cells to obtain a yeast crude extract; and

(iv) and carrying out solid-liquid separation on the yeast crude extract to obtain a liquid part, namely the yeast cell extract.

In another preferred embodiment, the solid-liquid separation comprises centrifugation.

In another preferred embodiment, the centrifugation is carried out in the liquid state.

In another preferred embodiment, the centrifugation conditions are 5000-.

In another preferred embodiment, the centrifugation time is 0.5-2h, preferably 20min-50 min.

In another preferred embodiment, the centrifugation is carried out at 1-10 ℃, preferably at 2-6 ℃.

In another preferred embodiment, the washing treatment is carried out using a washing solution at a pH of 7 to 8 (preferably, 7.4).

In another preferred embodiment, the washing solution is selected from the group consisting of: potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

In another preferred example, the cell disruption treatment comprises high-pressure disruption, freeze-thawing (such as liquid nitrogen low temperature) disruption.

In a fourth aspect, the invention provides an in vitro cell-free protein synthesis system comprising a cell extract according to the third aspect of the invention.

In another preferred embodiment, the protein synthesis system consists of or consists essentially of a cell extract according to the third aspect of the invention.

In another preferred embodiment, the cell-free protein synthesis system further comprises one or more components selected from the group consisting of:

(a) a cell extract;

(b) a substrate for synthesizing a protein;

(c) a substrate for RNA synthesis;

(d) does not contain or contains RNA polymerase.

In another preferred embodiment, the synthesis system further comprises one or more components selected from the group consisting of: magnesium ions, potassium ions, buffers, energy regeneration systems, polyethylene glycol, glucose, phosphate, Dithiothreitol (DTT) and optionally a solvent, sucrose, said solvent being water or an aqueous solvent.

In another preferred embodiment, the substrate for synthesizing RNA comprises: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof.

In another preferred embodiment, the substrate of the synthetic protein comprises: 1-20 kinds of natural amino acids and non-natural amino acids.

In another preferred embodiment, the magnesium ion is derived from a magnesium ion source selected from the group consisting of: magnesium acetate, magnesium glutamate, or a combination thereof.

In another preferred embodiment, the phosphate is selected from the group consisting of: potassium phosphate, monopotassium phosphate, sodium phosphate, ammonium phosphate, monosodium phosphate, disodium phosphate, dipotassium phosphate, or combinations thereof.

In another preferred embodiment, the potassium ion is derived from a potassium ion source selected from the group consisting of: potassium acetate, potassium glutamate, or a combination thereof.

In another preferred embodiment, the energy regeneration system is selected from the group consisting of: a phosphocreatine/phosphocreatine enzyme system, a glycolytic pathway and its intermediate energy system, or a combination thereof.

In another preferred embodiment, the cell-free protein synthesis system further comprises an artificially synthesized tRNA.

In another preferred embodiment, the buffer is selected from the group consisting of: 4-hydroxyethylpiperazine ethanesulfonic acid, tris, or a combination thereof.

In another preferred embodiment, the cell-free protein synthesis system further comprises an exogenous DNA molecule for directing protein synthesis.

In another preferred embodiment, the DNA molecule is linear.

In another preferred embodiment, the DNA molecule is circular.

In another preferred embodiment, the DNA molecule comprises a sequence encoding a foreign protein.

In another preferred embodiment, the sequence encoding the foreign protein includes a genomic sequence and a cDNA sequence.

In another preferred embodiment, the sequence encoding the foreign protein further comprises a promoter sequence, a 5 'untranslated sequence, and a 3' untranslated sequence.

In another preferred embodiment, the cell-free protein synthesis system comprises a component selected from the group consisting of: 4-hydroxyethylpiperazine ethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphates, amino acids, phosphocreatine, Dithiothreitol (DTT), phosphocreatine kinase, RNA polymerase, or a combination thereof.

In another preferred embodiment, the polyethylene glycol is selected from the group consisting of: PEG3000, PEG8000, PEG6000, PEG3350, or combinations thereof.

In another preferred embodiment, the polyethylene glycol comprises polyethylene glycol with molecular weight (Da) of 200-.

In another preferred embodiment, the nucleoside triphosphate is selected from the group consisting of: adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate, uracil nucleoside triphosphate, or combinations thereof.

In another preferred embodiment, the amino acid is selected from the group consisting of: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof.

In another preferred embodiment, the amino acids include D-form amino acids and/or L-form amino acids.

In another preferred embodiment, the concentration of potassium acetate in the protein synthesis system is 20-210mM, preferably 30-150mM, more preferably 30-60 mM.

In another preferred embodiment, the concentration of magnesium acetate in the protein synthesis system is 1-10mM, preferably 1-5mM, more preferably 2-4 mM.

In another preferred embodiment, the concentration of said creatine phosphate in said protein synthesis system is 10-50mM, preferably 20-30mM, more preferably 25 mM.

In another preferred embodiment, the concentration of Dithiothreitol (DTT) in the protein synthesis system is 0.2-15mM, preferably 0.2-7mM, more preferably 1-2 mM.

In another preferred embodiment, the concentration of phosphocreatine kinase in the protein synthesis system is 0.1-1mg/mL, preferably 0.2-0.5mg/mL, more preferably 0.27 mg/mL.

In another preferred embodiment, in the protein synthesis system, the RNA polymerase is T7RNA polymerase, and the concentration of the T7RNA polymerase is 0.01-0.3mg/mL, preferably 0.02-0.1mg/mL, and more preferably 0.027-0.054 mg/mL.

In a fifth aspect, the present invention provides a method for in vitro protein synthesis, comprising the steps of:

(i) providing an in vitro cell-free protein synthesis system according to the fourth aspect of the invention, and adding exogenous DNA molecules for directing protein synthesis;

(ii) (ii) incubating the protein synthesis system of step (i) under suitable conditions for a period of time to synthesize a protein encoded by the exogenous DNA.

In another preferred example, the method further comprises: (iii) optionally isolating or detecting said protein encoded by the foreign DNA from said protein synthesis system.

In another preferred embodiment, the exogenous DNA is from a prokaryote or a eukaryote.

In another preferred embodiment, the exogenous DNA is from an animal, plant, pathogen.

In another preferred embodiment, the exogenous DNA is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.

In another preferred embodiment, the coding sequence for the exogenous protein encodes an exogenous protein selected from the group consisting of a luciferin protein, or a luciferase (e.g., firefly luciferase), a green fluorescent protein, a yellow fluorescent protein, an aminoacyl tRNA synthetase, a glyceraldehyde-3-phosphate dehydrogenase, a catalase, an actin, a variable region of an antibody, a luciferase mutant, α -amylase, enteromycin A, hepatitis C virus E2 glycoprotein, an insulin precursor, interferon α A, interleukin-1 β, lysozyme, serum albumin, a single chain antibody fragment (scFV), a transthyretin, a tyrosinase, a xylanase, or a combination thereof.

In another preferred embodiment, the exogenous protein is selected from the group consisting of a luciferin protein, or a luciferase enzyme (e.g., firefly luciferase), a green fluorescent protein, a yellow fluorescent protein, an aminoacyltRNA synthetase, a glyceraldehyde-3-phosphate dehydrogenase, a catalase, an actin, a variable region of an antibody, a luciferase mutation, α -amylase, enteromycin A, hepatitis C virus E2 glycoprotein, an insulin precursor, interferon α A, interleukin-1 β, lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.

In another preferred embodiment, the exogenous DNA encodes an exogenous protein selected from the group consisting of a luciferin protein, or a luciferase (e.g., firefly luciferase), a green fluorescent protein, a yellow fluorescent protein, an aminoacyl tRNA synthetase, a glyceraldehyde-3-phosphate dehydrogenase, a catalase, an actin, a variable region of an antibody, a luciferase mutant, α -amylase, enteromycin A, hepatitis C virus E2 glycoprotein, an insulin precursor, interferon α A, interleukin-1 β, a lysozyme, serum albumin, a single chain antibody fragment (scFV), a transthyretin, a tyrosinase, a xylanase, or a combination thereof.

In another preferred embodiment, the exogenous DNA encodes a protein selected from the group consisting of a luciferin protein, or a luciferase (such as firefly luciferase), a green fluorescent protein, a yellow fluorescent protein, an aminoacyltRNA synthetase, a glyceraldehyde-3-phosphate dehydrogenase, a catalase, an actin, a variable region of an antibody, a luciferase mutation, α -amylase, an enteromycin A, hepatitis C virus E2 glycoprotein, an insulin precursor, interferon α A, interleukin-1 β, a lysin, a serum albumin, a single chain antibody fragment (scFV), a transthyretin, a tyrosinase, a xylanase, or a combination thereof.

However, the above is only an exemplary introduction of the foreign protein, and does not include only the above foreign protein; can be any protein capable of being exogenously expressed.

In another preferred embodiment, in the step (ii), the reaction temperature is 20 to 37 ℃, preferably 20 to 25 ℃.

In another preferred embodiment, in the step (ii), the reaction time is 1 to 72 hours, preferably 2 to 23 hours.

The sixth aspect of the invention provides an in vitro protein synthesis system, which at least comprises a cell lysate, wherein the cell lysate comes from a genetic engineering cell, and a polynucleotide sequence constructed by inserting a strong promoter sequence in front of a nucleotide sequence for encoding eIF4E binding protein is integrated in a gene sequence of the genetic engineering cell and is used for improving the expression amount of a foreign protein in the in vitro protein synthesis system; and no or additional eIF4E binding protein was added to the synthesis system.

Equivalently, there is provided an in vitro protein synthesis system for increasing the expression level of a foreign protein, comprising:

(a) a cell lysate;

(b) none or a substrate for RNA synthesis;

(c) a substrate for synthesizing a protein;

(d) no or containing RNA polymerase;

(e) no or additional eIF4E binding protein;

wherein, the cell lysate is the lysate of cells containing a polynucleotide sequence or a vector constructed by inserting a strong promoter sequence in front of a nucleotide sequence of the eIF4E binding protein.

In another preferred embodiment, the strong promoter sequence is selected from one of GAPDH1, HXK1, PGK1, TEF1, TIF1, ADH1, SED1, or a combination thereof.

In another preferred embodiment, the strong promoter sequence is selected from one of pScGAPDH1, pKlHXK1, pKlPGK1, pScPGK1, pScSED1, pscaldh 1, pKlGAPDH1 or combinations thereof.

In another preferred example, the eIF4E binding protein is one of EAP1p, p20 from yeast, 4E-BP1, 4E-BP2 from human, PHAS-I from mouse, or their combination.

In another preferred embodiment, the cell is a eukaryotic cell.

In another preferred embodiment, the cell is one of a mammalian cell, a plant cell, a yeast cell, an insect cell or any combination thereof.

In another preferred embodiment, the cell is one of yeast, wheat germ cell, rabbit reticulocyte, or any combination thereof.

In another preferred embodiment, the yeast is selected from one of saccharomyces cerevisiae, kluyveromyces yeast or a combination thereof.

In another preferred embodiment, the yeast of the genus kluyveromyces is selected from one of kluyveromyces lactis, kluyveromyces marxianus, kluyveromyces polybubali, or any combination thereof.

In another preferred embodiment, the cell lysate is an essential component for forming an in vitro protein synthesis system, and may be a liquid cell lysate, a lyophilized powder obtained by freeze-drying a cell lysate, or other forms. In the case of lyophilized powder, the lyophilized powder is reconstituted with a buffer commonly used in vitro synthesis systems (including but not limited to 4-hydroxyethylpiperazineethanesulfonic acid, tris, or a combination thereof). The cell lysate contains eIF4E binding protein, and is used for improving the expression of foreign protein in an in vitro protein synthesis system.

In another preferred example, the cell lysate may itself contain eIF4E binding protein expressed endogenously by the cell, i.e., the cell itself contains the gene encoding the eIF4E binding protein, and expression of the eIF4E binding protein is enhanced by inserting a strong promoter upstream of the coding sequence for the eIF4E binding protein.

In another preferred example, the cell lysate may not contain the endogenous eIF4E binding protein, i.e., the cell itself does not contain the gene encoding the eIF4E binding protein, but is modified to incorporate a polynucleotide sequence or vector constructed by inserting a strong promoter sequence in front of the nucleotide sequence encoding the eIF4E binding protein, to achieve high expression of the eIF4E binding protein.

In another preferred embodiment, the eIF4E-binding protein contained in the cell lysate may be homologous or heterologous. For example, a strong promoter may be inserted directly upstream of the coding sequence for the EAP1p protein (KlEAP1p) in kluyveromyces lactis to enhance expression of the eIF4E binding protein; or a polynucleotide sequence constructed by inserting a strong promoter sequence in front of the nucleotide sequence coding EAP1p protein (ScEAP1p) of the saccharomyces cerevisiae can be integrated in the kluyveromyces lactis to enhance the expression of the ScEAP1p protein; the expression of the 4E-BP1 and 4E-BP2 proteins can also be enhanced by inserting a polynucleotide sequence constructed by a strong promoter sequence in front of the nucleotide sequence encoding the human 4E-BP1 and 4E-BP2 proteins into yeast cells.

In another preferred embodiment, the cell lysate may be derived from a single cell, or may be derived from the lysate of two or more cells. When derived from lysates of two or more cells, eIF4E-binding protein may be overexpressed in all cells, or in several of them, but in at least one of them.

In another preferred embodiment, eIF4E binding protein may be additionally added to the in vitro protein synthesis system, whether or not the cells are engineered to contain over-expressed eIF4E binding protein in the cell lysate. The same technical effect can be obtained by additionally adding the eIF4E binding protein even if cells which are not modified by expressing the eIF4E binding protein are used. Additional eIF4E binding proteins may be obtained by expression in other cells or cell-free in vitro synthesis, with or without purification.

In another preferred example, the in vitro protein synthesis system may contain one or more eIF4E binding proteins that are present in the cell lysate by endogenous expression of the cell and/or by additional addition of eIF4E binding proteins.

The seventh aspect of the present invention provides a method for in vitro synthesis of a protein, the method comprising the steps of:

(i) providing a protein synthesis system, wherein said synthesis system is the synthesis system according to the sixth aspect of the present invention; and

(ii) incubating said in vitro protein synthesis system in the presence of a DNA or RNA template encoding said foreign protein under conditions suitable for expression of the protein, thereby expressing said foreign protein.

In another preferred example, the method further comprises: (iii) isolating or detecting the foreign protein.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a plasmid map of pKM-CAS1.0-KlURA 3.

FIG. 2 shows the results of in vitro translation activity assay.

Detailed Description

After extensive and intensive studies, a novel genetically engineered strain is obtained by first screening through a large number of screens and grops, wherein a first exogenous gene expression cassette expressing a first nucleic acid construct consisting of coding sequences of promoter elements (such as pScGAPDH1, pScHXK1, pScPGK1, pScTEF1, pScTIF1, pScADH1, pScTEF1 and other strong promoter elements, wherein p is a short hand of promoter, is a promoter, Sc is a Saccharomyces cerevisiae source, Kluyveromyces is a source, and subsequent letters are gene names of corresponding promoters) and EAP1p (such as KlEAP1p) proteins is integrated in the genome of the strain, and the efficiency of protein translation is remarkably improved by enhancing the expression or activity of the KlEAPlp (such as EAP1p) protein in the strain. On this basis, the present inventors have completed the present invention.

Term(s) for

As used herein, the terms "engineered strain" and "genetically engineered strain" are used interchangeably and refer to the engineered strain of the invention for use in increasing the efficiency of in vitro protein synthesis, i.e., the strain of the first aspect of the invention.

As used herein, the term "cell lysate" may be a cell lysate, or may be other morphological products formed after the cell lysate is processed, such as lyophilized powder.

As used herein, the terms "cell lysate", "cell extract" are used interchangeably.

eIF4E binding proteins

Eukaryotic initiation factor 4E (eukaryotic initiation factor 4E, abbreviated as eIF 4E) is a cap binding protein, is a key regulation target point in the eukaryotic protein synthesis pathway, can specifically recognize the cap structure at the 5' end of mRNA, and plays an important role in the initiation process of eukaryotic translation. In eukaryotes, eIF4E binding proteins include Eap1p, p20, etc., of yeast origin, and also include 4E-BP1, 4E-BP2 of human origin, PHAS-I of rabbit origin, and 4E-BP1, 4E-BP2 of human origin, and PHAS-I of rabbit origin.

Among them, Eap1p (eIF4E-associated protein 1) plays an important role in yeast cell growth and can inhibit in vitro cap-dependent translation by binding to eIF 4E. p20(Caf20p) also performs the same function.

Based on the EAP1 gene sequence in Saccharomyces cerevisiae, EAP1 gene is used in BLAST comparison analysis in UniProt database to determine the nucleotide sequence and corresponding protein sequence of EAP1 homologous gene in Kluyveromyces lactis.

In a preferred embodiment, the nucleotide sequence of the KlEAP1 is shown in SEQ ID No. 1.

ATGTCCGACACAGCAGAAGAGAATGGCCTTACGAGGTTTCTTATGAAGGTGAAGGAAAATCTGGATACCAGCCAGTCAGAAGCTCAAACTGAGACCGAACCCATTGAATTTAAGTACACTTTTGATTATAAGCCTGAATTCAGCAGTGGCAAGGTGGTCTATTCACGGAACGAATTGCTTGCTATTCGCGAGCAAGTTGCAGAGGAAGACGTGACCAATCTAGCTTCTGAATTGCCTAACAAGAAGTTTTGGAGATTACCCGTTCCAGGATCCAACGTCGGTGGTAGGAAGGGATCCAACACCAGAGGTAACCATGATGATAAGTTTGGCGGAAAGGACAGAGGTGCCCAGAGTGGTAGCAGAAATGCCAGGAACAATAGAAATAGCAAGCGTCAAGGTGGTAAAAAGGCTGGGAAGGAGAGCAACGAAGAATACATTGCTTTGGAAGAACAGATGGAATCTACAGGGAATCCAATGGCAGATTTTGAGAATTGGAGAAACAAAATGAAGGAACTGGAGCGTCAAAAAAGAGGGTTGGACTCTGATGCGGGCAAAGATTCGGATTCTCCTGCAGGATTGCCAGCTCAAAGTTTCAGCTCCATATCCGATTTCTTCAATTTAAAGCCGGATGACCAAAAGAGTGCTCCATTGGAAGAGCTTGAGCCAGCGGACTCGTCGGAAGACGTTTCGAAACAAACATTTGAAAAACAAGATAGAGATTCCCAAGATGTTCAACACGGCAGTCAAGGCCAAGGCATAAGCAAGAGCAATTCTTCCAGATTCTCCTCGTTCTTCCAAGGTGGTAATTCTCCGGATGCCAGTGATAACCGTCCTATCGCGAAGCCTCCAAGTCAATCTTCGGAAGATAGCAGACCAGCTGCGGGATCCAGGCTACTCTCTCTATTCAATACTGACTCACCATCTTCAGATTCGGTAGTTCAACACCAACAACCTGAAAAGCCCATGGTAAACAATCCACCTGGTCTTACTCAACAGTCTTCTACCACATCATTGTCGTCGGTCGCTTCTTCGAATCATTCCCAACCTCACTCAGGTCCTCGTGCTGCGAAAGATAATGATGTCAATGGCAGCGTGTTTTTGAAGAGTTTAATGACTAAAGGCAGTGAAAATATGATGCAACAGCCCTTAGTCTCTGCCCCTCCGGGCCTTTCCCAAAATCAATCTCAGATACCTAACATCACACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCATCAGCAACAGCAACAACAGCAACAACAGCAACACATTCAGCGCCCACATCAACAGCAGCAGCAGCAACCTCAACAACGCAAACTTCAACAAATTCATCAACAACACAGCCAGCATGCTCATCCTGCTCAGCAGCAGCAACATGGTAAACCACCACAAAACATTGCTCAATCAGGTGCTCCAATGCAGGCACCTCCAGGATTCCCAGTTGGGATGCCACCACATATGATGGCCCCTCCTATGGGAATGCCTCCACAGCACTTCCAAGGTGGCTATGCTATGCCTCCTCCACCGCCACCTGGAATGGGACAGCCACGTATAATGAGCAACGGTAAAGGATTCCCCGAACACTTGGGTAACCCACAACAGCAGCCTCCTAGCGCTCCAAAACAGCCAGCAAGTAAAGTTGGTCCGCAAGGTCAAGTTCCTTCACAAGGCCATCCTCAATCCCACCCACAACAACCTTATATGATGTCAGGTATGCCAATGAACTTCAATGGGCAAAATGTTCCAATTCCAGCTCAAGGTATCCCACCAAATGCATTCCCATATGGCCATCCAATGATGGCTCTACAGTTTCAACAACAACAACAGCAGCAGCAGCAACAACAATACCCGCAGCAACAACAACACCCGCAGCAACCGCGCCAAACAAATCCGCGCCAATAA；

In a preferred embodiment, the protein sequence of the KlEAP1p is shown in SEQ ID No. 2.

MSDTAEENGLTRFLMKVKENLDTSQSEAQTETEPIEFKYTFDYKPEFSSGKVVYSRNELLAIREQVAEEDVTNLASELPNKKFWRLPVPGSNVGGRKGSNTRGNHDDKFGGKDRGAQSGSRNARNNRNSKRQGGKKAGKESNEEYIALEEQMESTGNPMADFENWRNKMKELERQKRGLDSDAGKDSDSPAGLPAQSFSSISDFFNLKPDDQKSAPLEELEPADSSEDVSKQTFEKQDRDSQDVQHGSQGQGISKSNSSRFSSFFQGGNSPDASDNRPIAKPPSQSSEDSRPAAGSRLLSLFNTDSPSSDSVVQHQQPEKPMVNNPPGLTQQSSTTSLSSVASSNHSQPHSGPRAAKDNDVNGSVFLKSLMTKGSENMMQQPLVSAPPGLSQNQSQIPNITQQQQQQQQQQQHQQQQQQQQQQHIQRPHQQQQQQPQQRKLQQIHQQHSQHAHPAQQQQHGKPPQNIAQSGAPMQAPPGFPVGMPPHMMAPPMGMPPQHFQGGYAMPPPPPPGMGQPRIMSNGKGFPEHLGNPQQQPPSAPKQPASKVGPQGQVPSQGHPQSHPQQPYMMSGMPMNFNGQNVPIPAQGIPPNAFPYGHPMMALQFQQQQQQQQQQQYPQQQQHPQQPRQTNPRQ。

Exogenous gene expression cassette

The term "exogenous gene expression cassette" as used herein refers to a cassette with a first exogenous gene that expresses a first nucleic acid construct.

The engineering strain is obtained by protoplast fusion of a recombinant strain integrated with a first exogenous gene expression cassette. The engineering strain is simultaneously integrated with a first exogenous gene expression cassette.

In vitro expression system

Yeast (yeast) combines the advantages of simple culture, efficient protein folding, and post-translational modification. Wherein, the Saccharomyces cerevisiae (Saccharomyces cerevisiae) and the Pichia pastoris (Pichia pastoris) are model organisms for expressing complex eukaryotic proteins and membrane proteins, and the yeast can also be used as a raw material for preparing an in vitro translation system.

Kluyveromyces (Kluyveromyces) is a species of ascosporogenous yeast, of which Kluyveromyces marxianus and Kluyveromyces lactis (Kluyveromyces lactis) are industrially widely used. In comparison with other yeasts, kluyveromyces lactis has many advantages such as superior secretion ability, better large-scale fermentation characteristics, a level of food safety, and the ability to modify proteins post-translationally.

In the present invention, the yeast in vitro expression system is not particularly limited, and a preferred yeast in vitro expression system is a Kluyveromyces expression system (more preferably, a Kluyveromyces lactis expression system).

The in vitro expression system is not limited to yeast, more specifically to a Kluyveromyces lactis expression system, but the examples are only illustrative of the system, and other eukaryotic in vitro expression systems are also applicable.

Protein synthesis system

An in vitro protein synthesis system, which at least comprises a cell lysate, wherein the cell lysate is from a genetic engineering cell, and a polynucleotide sequence constructed by inserting a strong promoter sequence in front of a nucleotide sequence for encoding eIF4E binding protein is integrated in a gene sequence of the genetic engineering cell and is used for improving the expression level of a foreign protein in the in vitro protein synthesis system; and no or additional eIF4E binding protein was added to the synthesis system.

(a) a cell lysate;

(b) none or a substrate for RNA synthesis;

(c) a substrate for synthesizing a protein;

(d) no or containing RNA polymerase;

(e) no or additional eIF4E binding protein;

In another preferred embodiment, the cell is a eukaryotic cell.

In another preferred embodiment, the cell is a yeast, wheat germ cell.

Specifically, for the components of an in vitro cell-free protein synthesis system, the synthesis system comprises:

(a) a cell extract;

(b) a substrate for synthesizing a protein;

(c) a substrate for RNA synthesis;

(d) does not contain or contains RNA polymerase.

In another preferred embodiment, the synthesis system further comprises one or more components selected from the group consisting of: magnesium ions, potassium ions, buffers, energy regeneration systems, polyethylene glycol, glucose, phosphate, Dithiothreitol (DTT), and optionally a solvent, which is water or an aqueous solvent.

In a particularly preferred embodiment, the in vitro protein synthesis system provided by the present invention comprises: yeast cell extract, 4-hydroxyethylpiperazine ethanesulfonic acid, potassium acetate, magnesium acetate, adenine nucleoside triphosphate (ATP), guanine nucleoside triphosphate (GTP), cytosine nucleoside triphosphate (CTP), thymidylate nucleoside triphosphate (TTP), amino acid mixture, phosphocreatine, Dithiothreitol (DTT), phosphocreatine kinase, luciferase DNA, RNA polymerase.

In the present invention, the RNA polymerase is not particularly limited and may be selected from one or more RNA polymerases, and a typical RNA polymerase is T7RNA polymerase.

In the present invention, the proportion of the yeast cell extract in the in vitro protein synthesis system is not particularly limited, and usually the yeast cell extract accounts for 20 to 70%, preferably 30 to 60%, more preferably 40 to 50% of the in vitro protein synthesis system.

In the present invention, the yeast cell extract does not contain intact cells, and typical yeast cell extracts include ribosomes for protein translation, transfer RNAs, aminoacyl tRNA synthetases, initiation and elongation factors required for protein synthesis, and termination and release factors. In addition, the yeast extract also contains some other proteins, especially soluble proteins, which originate in the cytoplasm of the yeast cell.

In the present invention, the yeast cell extract contains 20 to 100mg/ml, preferably 50 to 100mg/ml of protein. The method for determining the protein content is a Coomassie brilliant blue determination method.

In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method comprises the steps of:

(i) providing a yeast cell;

(ii) washing the yeast cells to obtain washed yeast cells;

(iii) performing cell breaking treatment on the washed yeast cells to obtain a yeast crude extract;

In the present invention, the solid-liquid separation method is not particularly limited, and a preferable method is centrifugation.

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

In the present invention, the centrifugation conditions are not particularly limited, and one preferable centrifugation condition is 5000-.

In the present invention, the centrifugation time is not particularly limited, and a preferable centrifugation time is 0.5min to 2h, preferably 20min to 50 min.

In the present invention, the temperature of the centrifugation is not particularly limited, and it is preferable that the centrifugation is performed at 1 to 10 ℃, preferably, 2 to 6 ℃.

In the present invention, the washing treatment is not particularly limited, and a preferable washing treatment is a treatment with a washing solution at a pH of 7 to 8 (preferably, 7.4), the washing solution is not particularly limited, and typically the washing solution is selected from the group consisting of: potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

In the present invention, the manner of the cell disruption treatment is not particularly limited, and a preferable cell disruption treatment includes high-pressure disruption, freeze-thawing (e.g., liquid nitrogen low-temperature disruption).

The nucleoside triphosphate mixture in the in vitro protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate. In the present invention, the concentration of each mononucleotide is not particularly limited, and usually the concentration of each mononucleotide is 0.5 to 5mM, preferably 1.0 to 2.0 mM.

The amino acid mixture in the in vitro protein synthesis system may comprise natural or unnatural amino acids, and may comprise D-or L-amino acids. Representative amino acids include (but are not limited to) the 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The concentration of each amino acid is usually 0.01-0.5mM, preferably 0.02-0.2mM, such as 0.05, 0.06, 0.07, 0.08 mM.

In a preferred embodiment, the in vitro protein synthesis system further comprises polyethylene glycol or an analog thereof. The concentration of polyethylene glycol or an analog thereof is not particularly limited, and usually, the concentration (w/v) of polyethylene glycol or an analog thereof is 0.1 to 8%, preferably 0.5 to 4%, more preferably 1 to 2%, based on the total weight of the protein synthesis system. Representative PEG examples include (but are not limited to): PEG3000, PEG8000, PEG6000 and PEG 3350. It is understood that the systems of the present invention may also include other polyethylene glycols of various molecular weights (e.g., PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).

In a preferred embodiment, the in vitro protein synthesis system further comprises sucrose. The concentration of sucrose is not particularly limited, and generally, the concentration of sucrose is 0.03 to 40 wt%, preferably 0.08 to 10 wt%, more preferably 0.1 to 5 wt%, based on the total weight of the protein synthesis system.

A particularly preferred in vitro protein synthesis system comprises, in addition to yeast extract, the following components: 22mM of 4-hydroxyethylpiperazine ethanesulfonic acid with the pH value of 7.4, 30-150mM of potassium acetate, 1.0-5.0mM of magnesium acetate, 1.5-4mM of nucleoside triphosphate mixture, 0.08-0.24mM of amino acid mixture, 25mM of phosphocreatine, 1.7mM of dithiothreitol, 0.27mg/mL of phosphocreatine kinase, 1% -4% of polyethylene glycol, 0.5% -2% of sucrose, 8-20ng/μ l of DNA of firefly luciferase and 0.027-0.054mg/mL of T7RNA polymerase.

The main advantages of the invention include:

(i) the present invention first constructs a nucleic acid construct incorporating a coding sequence for a strong promoter element, such as the proteins pScGAPDH1, pScPGK1, pScTEF1, pScTIF1, pScADH1, pScTEF1, pKLADH1, pKLGPADH1, pKLHXK1, pKLPGK1, pKLTEF1, pKLTIF1 and KlEAP1 p; and engineered strains comprising the nucleic acid constructs.

(ii) The invention discovers for the first time that the cell extract (such as yeast cell extract) derived from the engineering strain can obviously improve the efficiency of the in vitro protein synthesis system for producing protein.

(iii) The invention discovers for the first time that the strain has stronger in vitro protein synthesis capacity than that of a wild type bacterium group, the relative fluorescence unit value emitted by the coded and synthesized eGFP protein reaches 110.5, while the relative fluorescence unit value of the eGFP protein synthesized by the wild type yeast strain is only 12.3, which is improved by about 8.98 times.

(iv) The invention can realize the overexpression of the EAP1p protein, thereby obviously improving the efficiency of generating the protein by an in vitro protein synthesis system.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

Example 1 insertion of a Strong promoter before KlEAP1 Gene by CRISPR-Cas9 technology

1.1KlURA3 sequence search and CRISPR gRNA sequence determination

The URA3 gene is a gene on the V chromosome of yeast, can perform positive selection and negative selection, and is one of the most important genetic markers in yeast genetic engineering. Examples URA3 was chosen as a selection marker, although other commonly used selection markers (genetic markers) may be selected.

Based on the URA3 gene sequence in Saccharomyces cerevisiae (Saccharomyces cerevisiae), BLAST alignment analysis was performed with URA3 gene in the UniProt database to determine the URA3 homologous gene sequence in Kluyveromyces lactis, which was named KlURA3 (2034995.. 2035798 located on chromosome E). At the KlURA3 gene stop codon, adjacent PAM sequences (NGG) were searched and gRNA sequences were determined. The principle of gRNA selection is: the GC content is moderate and is 40-60 percent; avoiding the presence of poly T structures. The KlURA3gRNA sequence identified was GCGCTCCCCATTAATTATAC, located at position 424927.. 424936 of chromosome E. The tail of the gene is inserted with a piece of marker DNA as an example, and other target genes or insertion positions and sequences can be operated by a similar method.

1.2CRISPR-Cas9 mediated construction of plasmid with strong promoter inserted before KlEAP1

To achieve overexpression of klep 1p, the promoters of pscaldh 1, pScGAPDH1, pScPGK1, pScSED1, pScTEF1, pScTIF1 and pKlGAPDH1, pklhxkk 1, pKlPGK1, pKlTEF1, pKlTIF1 were inserted before the klep 1 gene by CRISPR-Cas technology, respectively. The plasmid construction and transformation method is as follows:

1.2.1CRISPR plasmid construction

PCR amplification was carried out using primers PF1: CTTGAAACAAGGTGCGCAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT, PR1: CTTGCGCACCTTGTTTCAAGTGCATCGGCCGGGAATCGAACCCGGGGCCC, using pCas plasmid as template, mixing 17. mu.L of the amplified products, adding 1. mu.L of DpnI, 2. mu.L of 10 Xdigestion buffer, 3h of warm bath at 37 ℃, adding 10. mu.L of the product after treatment with DpnI into 100. mu.L of DH5 α competent cells, placing on ice for 30min, after heat shock 45s at 42 ℃, adding 1mLLB liquid medium, shaking for 1h at 37 ℃, coating on Kan-resistant LB solid culture, inverting for culture at 37 ℃ until single clone grows out, picking 5 single clones, shaking for culture in LB liquid medium, detecting positive PCR and confirming the sequencing, extracting plasmid for storage, named pKM-CAS1.0-KlURA3 (FIG. 1).

1.2.2 amplification of Donor DNA fragments

In order to rapidly amplify the linear donor DNA fragment, the linear donor DNA sequence was amplified by OVER-LAP PCR.

The genes of saccharomyces cerevisiae ADH1, GAPDH1, PGK1, SED1, TEF1 and TIF1 are respectively searched in a UniProt database, and the promoter sequences of ADH1, GAPDH1, PGK1, SED1, TEF1 and TIF1 are respectively named as pScADH1, pScGAPDH1, pScPGK1, pScSED1, pScTEF1 and pScTIF1 by using the upstream 1000bp of the initiation codon.

Based on the ADH1, GAPDH1, HXK1, PGK1, TEF1 and TIF1 gene sequences in Saccharomyces cerevisiae, the ADH1, GAPDH1, HXK1, PGK1, TEF1 and TIF1 genes are subjected to BLAST alignment analysis in the UniProt database, so as to determine the ADH1, GAPDH1, HXK1, PGK1, TEF1 and TIF1 homologous gene sequences in Kluyveromyces lactis, and the promoter sequences of the promoter of.

Based on the EAP1 gene sequence in Saccharomyces cerevisiae, BLAST alignment analysis is carried out with EAP1 gene in UniProt database to determine the EAP1 homologous gene sequence in Kluyveromyces lactis, which is named as KlEAP1 (located at 1231530..1233434 of chromosome B).

(1) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively as follows: f: ACACATTACTTGCCTCGAGCAT, respectively; r: GTACACCCGGAAACAACAAAAGGATTTAATGGGGAGCGCTGATTCTCTTT, and F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACAAATCAC, respectively; r: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

the ADH1 promoter sequence is amplified by taking saccharomyces cerevisiae genome DNA as a template, and the primer F: AAAGAGAATCAGCGCTCCCCATTAAATCCTTTTGTTGTTTCCGGGTGTAC, respectively; r: CATTCTCTTCTGCTGTGTCGGACATAGTTGATTGTATGCTTGGTATAGCT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: AGCTATACCAAGCATACAATCAACTATGTCCGACACAGCAGAAGAGAATG, respectively; r: GTGATTTGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pScADH1-KlEAP 1-DD.

(2) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: TTTGAAATGGCAGTATTGATAATGATTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACAAATCAC and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

the GPADH1 promoter sequence is amplified by taking saccharomyces cerevisiae genome DNA as a template, and the primer F: AAAGAGAATCAGCGCTCCCCATTAATCATTATCAATACTGCCATTTCAAA and R: CATTCTCTTCTGCTGTGTCGGACATTTTGTTTGTTTATGTGTGTTTATTC performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: GAATAAACACACATAAACAAACAAAATGTCCGACACAGCAGAAGAGAATG and R: GTGATTTGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pScGPADH1-KlEAP 1-DD.

(3) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: CACGAGTAATTCTTGCAAATGCCTATTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACAAATCAC and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

the PGK1 promoter sequence is amplified by taking saccharomyces cerevisiae genome DNA as a template, and the primer F: AAAGAGAATCAGCGCTCCCCATTAATAGGCATTTGCAAGAATTACTCGTG and R: CATTCTCTTCTGCTGTGTCGGACATTGTTTTATATTTGTTGTAAAAAGTA performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: TACTTTTTACAACAAATATAAAACAATGTCCGACACAGCAGAAGAGAATG and R: GTGATTTGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA 3-pScPGPK 1-KlEAP 1-DD.

(4) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, and respectively taking a primer F: ACACATTACTTGCCTCGAGCAT and R: TTGGGTGGAATGTTGTCGTTTTTCCTTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACAAATCAC and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

the SED1 promoter sequence is amplified by taking saccharomyces cerevisiae genome DNA as a template, and the primer F: AAAGAGAATCAGCGCTCCCCATTAAGGAAAAACGACAACATTCCACCCAA and R: CATTCTCTTCTGCTGTGTCGGACATCTTAATAGAGCGAACGTATTTTATT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: AATAAAATACGTTCGCTCTATTAAGATGTCCGACACAGCAGAAGAGAATG and R: GTGATTTGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pScSED1-KlEAP 1-DD.

(5) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: CAGACAAAATTCAATAAAGTTGCCTTTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACAAATCAC and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

the TEF1 promoter sequence is amplified by taking saccharomyces cerevisiae genome DNA as a template, and the primer F: AAAGAGAATCAGCGCTCCCCATTAAAGGCAACTTTATTGAATTTTGTCTG and R: CATTCTCTTCTGCTGTGTCGGACATCTTAGATTAGATTGCTATGCTTTCT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: AGAAAGCATAGCAATCTAATCTAAGATGTCCGACACAGCAGAAGAGAATG and R: GTGATTTGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pScTEF1-KlEAP 1-DD.

(6) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: ACAGAATTTTTCTGGAGCCAAATTGTTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACAAATCAC and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

and (2) amplifying a TIF1 promoter sequence by taking saccharomyces cerevisiae genome DNA as a template, and performing amplification by using a primer F: AAAGAGAATCAGCGCTCCCCATTAACAATTTGGCTCCAGAAAAATTCTGT and R: CATTCTCTTCTGCTGTGTCGGACATGATGAACTTTGCTCTATATTACACT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: AGTGTAATATAGAGCAAAGTTCATCATGTCCGACACAGCAGAAGAGAATG and R: GTGATTTGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pScTIF1-KlEAP 1-DD.

(7) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: ATGGCACACTGGTACTGCTTCGACTTTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACA and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify ADH1 promoter sequence, and taking a primer F: AAAGAGAATCAGCGCTCCCCATTAAAGTCGAAGCAGTACCAGTGTGCCAT and R: CATTCTCTTCTGCTGTGTCGGACATTTTATCTTTTTTTAGTATAGAGTTT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: AAACTCTATACTAAAAAAAGATAAAATGTCCGACACAGCAGAAGAGAATG and R: TGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pKLADH1-KlEAP 1-DD.

(8) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: TTCTGGCAGAAATGTGGTGTATGGGTTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACA and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

the sequence of the GAPDH1 promoter is amplified by taking Kluyveromyces lactis genome DNA as a template, and a primer F: AAAGAGAATCAGCGCTCCCCATTAACCCATACACCACATTTCTGCCAGAA and R: CATTCTCTTCTGCTGTGTCGGACATTTTATCTTTTTTTAGTATAGAGTTT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: ACATCAAAACAACAAATTAACAAAAATGTCCGACACAGCAGAAGAGAATG and R: TGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pKLGAPDH1-KlEAP 1-DD.

(9) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: TTTCCGGGCCCCGCTAGGTCTTTTTTTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACA and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify HXK1 promoter sequence, and taking a primer F: AAAGAGAATCAGCGCTCCCCATTAAAAAAAGACCTAGCGGGGCCCGGAAA and R: CATTCTCTTCTGCTGTGTCGGACATTCTTGATATTTATGTAATGTAATCT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: AGATTACATTACATAAATATCAAGAATGTCCGACACAGCAGAAGAGAATG and R: TGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pKLHXK1-KlEAP 1-DD.

(10) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: GTCTACGAGTGCTGAGGGCAGGCCCTTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACA and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

the sequence of a PGK1 promoter is amplified by taking Kluyveromyces lactis genome DNA as a template, and a primer F: AAAGAGAATCAGCGCTCCCCATTAAGGGCCTGCCCTCAGCACTCGTAGAC and R: CATTCTCTTCTGCTGTGTCGGACATTTTTATTAATTCTTGATCGATTTTT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: AAAAATCGATCAAGAATTAATAAAAATGTCCGACACAGCAGAAGAGAATG and R: TGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pKLPGK1-KlEAP 1-DD.

(11) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: TAAAGTGGTTTCATCGTGAAACCGTTTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACA and R: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

the genomic DNA of Kluyveromyces lactis is taken as a template to amplify a TEF1 promoter sequence, and a primer F: AAAGAGAATCAGCGCTCCCCATTAAACGGTTTCACGATGAAACCACTTTA and R: CATTCTCTTCTGCTGTGTCGGACATTTTTAATGTTACTTCTCTTGCAGTT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: AACTGCAAGAGAAGTAACATTAAAAATGTCCGACACAGCAGAAGAGAATG and R: TGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pKLTEF1-KlEAP 1-DD.

(12) Taking Kluyveromyces lactis genome DNA as a template to amplify a homologous arm sequence, wherein primers are respectively F: ACACATTACTTGCCTCGAGCAT and R: AGATACGTCTTCAACAATGTTGAACTTAATGGGGAGCGCTGATTCTCTTT, primer F: TGAGAAGGTTTTGGGACGCTCGAAGTTATACAGGAAACTTAATAGAACA and PR 2: CCTAACGGGATTTTCGCTTCGTGA performing PCR amplification;

the Kluyveromyces lactis genome DNA is used as a template to amplify the TIF1 promoter sequence, and a primer F: AAAGAGAATCAGCGCTCCCCATTAAGTTCAACATTGTTGAAGACGTATCT and R: CATTCTCTTCTGCTGTGTCGGACATCTTTACAGTTATGGATTTTCTAGTT performing PCR amplification;

taking Kluyveromyces lactis genome DNA as a template to amplify an EAP1 sequence, and taking a primer F: AACTAGAAAATCCATAACTGTAAAGATGTCCGACACAGCAGAAGAGAATG and R: TGTTCTATTAAGTTTCCTGTATAACTTCGAGCGTCCCAAAACCTTCTCA PCR amplification was performed.

The four amplification products were then diluted 50-fold and each pipetted 1.5. mu.L of each mix was used as template amplification donor sequence for PCR amplification with primers GCCAGCGTCAATACACTCCC and TTACGACAATGCCTAGTTGAGTGC. The obtained product is subjected to 1% agarose gel electrophoresis, recovered and purified to obtain donor DNA which is named as KlURA3-pKLTIF1-KlEAP 1-DD.

1.2.3 Kluyveromyces lactis transformation and Positive identification

Streaking the Kluyveromyces lactis liquid on a YPD solid culture medium, picking a single clone, carrying out shake culture in 25mL of 2 XYPD liquid culture medium overnight, and taking 2mL of the liquid to carry out shake culture in 50mL of the 2 XYPD liquid culture medium for 2-8 h. The yeast cells were collected by centrifugation at 3000g for 5min at 20 ℃ and resuspended in 500. mu.L of sterile water, and the cells were collected by centrifugation under the same conditions. A competent cell solution (5% v/v glycerol, 10% v/v DMSO) was prepared and yeast cells were dissolved in 500. mu.L of the solution. Subpackaging 50 μ L into 1.5mL centrifuge tubes, and storing at-80 deg.C.

Taking out 100 mu L of competent cells from a refrigerator at minus 80 ℃, thawing on ice, adding 200ng of gRNA & Cas9 plasmid (or gRNA/Cas9 fragment) and 1000ng of donor DNA fragment, uniformly mixing, transferring all into an electric shock cup, and carrying out ice bath for 2 min; 1.5kV, 200 omega, 25 muF electric shock, immediately adding 700 muL YPD, 30 ℃, shaking and incubating for 1-3 h at 200 rpm. The suspension was applied to a solid YPD (200. mu.g/mL G418) medium (200. mu.g/mL) in an amount of 200. mu.L, and cultured at 30 ℃ for 2 to 3 days until single colonies appeared. 10-20 monoclonals are picked from a plate transformed by the Kluyveromyces lactis, the plate is placed in 1mL YPD (200 mu G/mL G418) liquid culture medium for shaking culture overnight, and PCR detection is carried out on corresponding samples by taking a bacterial liquid as a template and a CRISPR insert Check primer pair. And determining the strains with positive PCR results and sequencing identification as positive strains.

1.3 in vitro translation Activity assay of the engineered Strain

The lactic acid Kluyveromyces strain after gene modification is prepared into an in-vitro protein synthesis system, and a green fluorescent protein gene DNA template is added to determine the protein translation capability of the modified strain. And (3) placing the reaction system in an environment with the temperature of 25-30 ℃, and standing and incubating for about 2-6 h. Immediately after the reaction, the reaction mixture was placed in an Envision 2120 multifunctional microplate reader (PerkinElmer), and read to detect the intensity of eGFP signal and Relative Fluorescence Unit (RFU) as an activity Unit.

As a control, PC is an unmodified wild yeast strain, which was prepared into an in vitro protein synthesis system, and protein translation ability was determined according to the same method; NC is that the DNA template of the firefly luciferase gene is not added in the prepared in-vitro protein synthesis system, and water with corresponding volume is added.

1.4 results

The results showed that 7 of the 12 engineered constructs showed a significant and significant improvement in protein synthesis capacity over the wild-type yeast cells, pKlPGK1:: KlEAP1, pScADH1:: KlEAP1, pKlGAPDH1:: KlEAP1, pKlHXK1:: KlEAP1, pScPGK1:: klep 1, pScSED1:: klep 1, pScGAPDH1:: klep 1, respectively. While the other 5 modified proteins had no or insignificant level of increase in synthesis capacity, ranging from 12.5 to 15.6.

The structure pKLGAPDH1 of a promoter pKLGAPDH1 is inserted in front of KlEAP1, and KlEAP1 shows stronger in vitro protein synthesis capacity than a wild-type yeast strain. The relative fluorescence unit value emitted by the coded and synthesized eGFP protein reaches 110.5, while the relative fluorescence unit value of the eGFP protein synthesized by the wild-type yeast strain is only 12.3, which is improved by about 8.98 times, and the fact that the KlEAP1 is modified can effectively enhance the protein synthesis efficiency of the yeast in-vitro protein synthesis system.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Reference to the literature

1.Sonenberg&Hinnebusch.,Regulation of translation initiation ineukaryotes:mechanisms and biological targets.Cell,2009.136(4):731-45.

2.Dever et al.,Mechanism and Regulation of Protein Synthesis inSaccharomyces cerevisiae.Genetics,2016.203(1):65-107.

3.M.,D.J.R.G.,Nucleic Acid.Encyclopedia of Cell Biology.Elsevier,2015.

4.Pause et al.,Insulin-dependent stimulation of protein synthesis byphosphorylation of a regulator of 5’-cap function.Nature,1994.371:762-767.

5.Gingras et al.,Regulation of 4E-BP1 phosphorylation:a novel two-step mechanism.Genes Dev,1999a.13:1422-1437.

6.Gingras et al.,Hierarchical phosphorylation of the translationinhibitor 4E-BP1.Genes Dev,2001.15:2852-2864.

7.Roux,P.P and Topisirovic,I.Regulation of mRNA translation bysignaling pathways.Cold Spring Harb Perspect Biol 2012.4:a012252

8.Adami,A.,et al.,Structure of TOR and Its Complex withKOG1.Molecular Cell,2007.27:p.509-516.

9.Cosentino,G.P.,et al.,Eap1p,a novel eukaryotic translationinitiation factor 4E-associated protein in Saccharomyces cerevisiae.2000.20:p.4604-4613.

10.Pause et al.,Insulin-dependent stimulation of protein synthesis byphosphorylation of a regulator of 5’-cap function.Nature,1994.371:762-767.

Sequence listing

<110> Kangma (Shanghai) Biotech Co., Ltd

<120> protein synthesis system for improving foreign protein expression level and application method thereof

<130>2018

<141>2018-09-25

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>1905

<212>DNA

<213> Kluyveromyces lactis (Kluyveromyces lactis)

<400>1

atgtccgaca cagcagaaga gaatggcctt acgaggtttc ttatgaaggt gaaggaaaat 60

ctggatacca gccagtcaga agctcaaact gagaccgaac ccattgaatt taagtacact 120

tttgattata agcctgaatt cagcagtggc aaggtggtct attcacggaa cgaattgctt 180

gctattcgcg agcaagttgc agaggaagac gtgaccaatc tagcttctga attgcctaac 240

aagaagtttt ggagattacc cgttccagga tccaacgtcg gtggtaggaa gggatccaac 300

accagaggta accatgatga taagtttggc ggaaaggaca gaggtgccca gagtggtagc 360

agaaatgcca ggaacaatag aaatagcaag cgtcaaggtg gtaaaaaggc tgggaaggag 420

agcaacgaag aatacattgc tttggaagaa cagatggaat ctacagggaa tccaatggca 480

gattttgaga attggagaaa caaaatgaag gaactggagc gtcaaaaaag agggttggac 540

tctgatgcgg gcaaagattc ggattctcct gcaggattgc cagctcaaag tttcagctcc 600

atatccgatt tcttcaattt aaagccggat gaccaaaaga gtgctccatt ggaagagctt 660

gagccagcgg actcgtcgga agacgtttcg aaacaaacat ttgaaaaaca agatagagat 720

tcccaagatg ttcaacacgg cagtcaaggc caaggcataa gcaagagcaa ttcttccaga 780

ttctcctcgt tcttccaagg tggtaattct ccggatgcca gtgataaccg tcctatcgcg 840

aagcctccaa gtcaatcttc ggaagatagc agaccagctg cgggatccag gctactctct 900

ctattcaata ctgactcacc atcttcagat tcggtagttc aacaccaaca acctgaaaag 960

cccatggtaa acaatccacc tggtcttact caacagtctt ctaccacatc attgtcgtcg 1020

gtcgcttctt cgaatcattc ccaacctcac tcaggtcctc gtgctgcgaa agataatgat 1080

gtcaatggca gcgtgttttt gaagagttta atgactaaag gcagtgaaaa tatgatgcaa 1140

cagcccttag tctctgcccc tccgggcctt tcccaaaatc aatctcagat acctaacatc 1200

acacagcagc agcagcagca gcagcagcag cagcagcatc agcaacagca acaacagcaa 1260

caacagcaac acattcagcg cccacatcaa cagcagcagc agcaacctca acaacgcaaa 1320

cttcaacaaa ttcatcaaca acacagccag catgctcatc ctgctcagca gcagcaacat 1380

ggtaaaccac cacaaaacat tgctcaatca ggtgctccaa tgcaggcacc tccaggattc 1440

ccagttggga tgccaccaca tatgatggcc cctcctatgg gaatgcctcc acagcacttc 1500

caaggtggct atgctatgcc tcctccaccg ccacctggaa tgggacagcc acgtataatg 1560

agcaacggta aaggattccc cgaacacttg ggtaacccac aacagcagcc tcctagcgct 1620

ccaaaacagc cagcaagtaa agttggtccg caaggtcaag ttccttcaca aggccatcct 1680

caatcccacc cacaacaacc ttatatgatg tcaggtatgc caatgaactt caatgggcaa 1740

aatgttccaa ttccagctca aggtatccca ccaaatgcat tcccatatgg ccatccaatg 1800

atggctctac agtttcaaca acaacaacag cagcagcagc aacaacaata cccgcagcaa 1860

caacaacacc cgcagcaacc gcgccaaaca aatccgcgcc aataa 1905

<210>2

<211>634

<212>PRT

<213> Kluyveromyces lactis (Kluyveromyces lactis)

<400>2

Met Ser Asp Thr Ala Glu Glu Asn Gly Leu Thr Arg Phe Leu Met Lys

1 5 10 15

Val Lys Glu Asn Leu Asp Thr Ser Gln Ser Glu Ala Gln Thr Glu Thr

20 25 30

Glu Pro Ile Glu Phe Lys Tyr Thr Phe Asp Tyr Lys Pro Glu Phe Ser

35 40 45

Ser Gly Lys Val Val Tyr Ser Arg Asn Glu Leu Leu Ala Ile Arg Glu

50 55 60

Gln Val Ala Glu Glu Asp Val Thr Asn Leu Ala Ser Glu Leu Pro Asn

65 70 75 80

Lys Lys Phe Trp Arg Leu Pro Val Pro Gly Ser Asn Val Gly Gly Arg

85 90 95

Lys Gly Ser Asn Thr Arg Gly Asn His Asp Asp Lys Phe Gly Gly Lys

100 105 110

Asp Arg Gly Ala Gln Ser Gly Ser Arg Asn Ala Arg Asn Asn Arg Asn

115 120 125

Ser Lys Arg Gln Gly Gly Lys Lys Ala Gly Lys Glu Ser Asn Glu Glu

130 135 140

Tyr Ile Ala Leu Glu Glu Gln Met Glu Ser Thr Gly Asn Pro Met Ala

145 150 155 160

Asp Phe Glu Asn Trp Arg Asn Lys Met Lys Glu Leu Glu Arg Gln Lys

165 170 175

Arg Gly Leu Asp Ser Asp Ala Gly Lys Asp Ser Asp Ser Pro Ala Gly

180 185 190

Leu Pro Ala Gln Ser Phe Ser Ser Ile Ser Asp Phe Phe Asn Leu Lys

195 200 205

Pro Asp Asp Gln Lys Ser Ala Pro Leu Glu Glu Leu Glu Pro Ala Asp

210 215 220

Ser Ser Glu Asp Val Ser Lys Gln Thr Phe Glu Lys Gln Asp Arg Asp

225 230 235 240

Ser Gln Asp Val Gln His Gly Ser Gln Gly Gln Gly Ile Ser Lys Ser

245 250 255

Asn Ser Ser Arg Phe Ser Ser Phe Phe Gln Gly Gly Asn Ser Pro Asp

260 265 270

Ala Ser Asp Asn Arg Pro Ile Ala Lys Pro Pro Ser Gln Ser Ser Glu

275 280 285

Asp Ser Arg Pro Ala Ala Gly Ser Arg Leu Leu Ser Leu Phe Asn Thr

290 295 300

Asp Ser Pro Ser Ser Asp Ser Val Val Gln His Gln Gln Pro Glu Lys

305 310 315 320

Pro Met Val Asn Asn Pro Pro Gly Leu Thr Gln Gln Ser Ser Thr Thr

325 330 335

Ser Leu Ser Ser Val Ala Ser Ser Asn His Ser Gln Pro His Ser Gly

340 345 350

Pro Arg Ala Ala Lys Asp Asn Asp Val Asn Gly Ser Val Phe Leu Lys

355 360 365

Ser Leu Met Thr Lys Gly Ser Glu Asn Met Met Gln Gln Pro Leu Val

370 375 380

Ser Ala Pro Pro Gly Leu Ser Gln Asn Gln Ser Gln Ile Pro Asn Ile

385 390 395 400

Thr Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Gln Gln Gln

405 410 415

Gln Gln Gln Gln Gln Gln Gln His Ile Gln Arg Pro His Gln Gln Gln

420 425 430

Gln Gln Gln Pro Gln Gln Arg Lys Leu Gln Gln Ile His Gln Gln His

435 440 445

Ser Gln His Ala His Pro Ala Gln Gln Gln Gln His Gly Lys Pro Pro

450 455 460

Gln Asn Ile Ala Gln Ser Gly Ala Pro Met Gln Ala Pro Pro Gly Phe

465 470 475 480

Pro Val Gly Met Pro Pro His Met Met Ala Pro Pro Met Gly Met Pro

485 490 495

Pro Gln His Phe Gln Gly Gly Tyr Ala Met Pro Pro Pro Pro Pro Pro

500 505 510

Gly Met Gly Gln Pro Arg Ile Met Ser Asn Gly Lys Gly Phe Pro Glu

515 520 525

His Leu Gly Asn Pro Gln Gln Gln Pro Pro Ser Ala Pro Lys Gln Pro

530 535 540

Ala Ser Lys Val Gly Pro Gln Gly Gln Val Pro Ser Gln Gly His Pro

545 550 555 560

Gln Ser His Pro Gln Gln Pro Tyr Met Met Ser Gly Met Pro Met Asn

565 570 575

Phe Asn Gly Gln Asn Val Pro Ile Pro Ala Gln Gly Ile Pro Pro Asn

580 585 590

Ala Phe Pro Tyr Gly His Pro Met Met Ala Leu Gln Phe Gln Gln Gln

595 600 605

Gln Gln Gln Gln Gln Gln Gln Gln Tyr Pro Gln Gln Gln Gln His Pro

610 615 620

Gln Gln Pro Arg Gln Thr Asn Pro Arg Gln

625 630

<210>3

<211>1200

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>3

agtcgaagca gtaccagtgt gccatagaga acgtgatgaa atggtaagtt atggtacgtg 60

atggaacacg ggaatagggg cgggacattt gggatctggt gaaagtgttg aattatcctt 120

caatagagga agtccgtgat tttctcttcc tgtccagtgg tttccagtgg gatcggtggc 180

tccactggaa gctggggaaa ccattgggtt tttgtcctct ttttagggac agagagttct 240

agggcatgga caatacgtta ccgagatgag ggacatccac tgttctgttc taacaaaaga 300

agatgaaatg attatcttcc ggactccagc ttttccattt gccttcgcgc ttgcctgtac 360

ggtcgttacc atacttacct ttcttgcttc ttctcaaaat ggaatccagg tttttagctt 420

cgtgtttctt tttttttttt accatttttc aaatttcagt cttccatttt cagcttcttg 480

tttttttttt tttttttttt tttcatttca gtttttacag cttccagtct ccctctttcc 540

ttcgatttcc atcttcttgt ttctgtagcc atcttcctat cacgtgcaag ggaagaaagt 600

gggggcccag caccttcttt tttgttcttt ggtgggggct cgttttagtt tttacgtgcg 660

taattgggaa tcttccgagg tagtatgaca tgtccggtgg tgacctgata ctttctgttt 720

ctccgagtag gacagaagag gaaaaaaaaa tagcgtgtga tgttcttcta ttctagtagt 780

gtattgatct attcacaatc agatcacaat catatgagca gatgatgtat tttgggttgc 840

ttttcaccaa cccaagtatt cgattgatct ttatatactg cggttatttc taggtcttaa 900

acggttaaca cctgttgcag ggtggtatgt atttttctca aagtgtgcta ttttcacacc 960

agctagaaat cagctgtctt acttgtatac aattagacca gccatttggt cttctggaat 1020

atgtatataa acacccggtc gattctgaca atccatccac ttttgtagta ggtctctcta 1080

tatccatttg tacaatgttg tttctgtttt gccctacatc atcatcaagc aaaaacaata 1140

gtttcaattg aaacataaac aagctttaaa cacacaaact ctatactaaa aaaagataaa 1200

<210>4

<211>1200

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>4

cccatacacc acatttctgc cagaatttca tgattacccg gccatgaatt ttccagcttc 60

cagttgtttt tcttctggaa atttcggctt ccggttaaaa aactaaaata actcaacatg 120

gaaagaaatt ggaatctgag gtttagtgga cgctgggatt ccactagagt cacaagctcc 180

ctagacatcc gaggacaact gacgaaactc tggcgctatt ttttccaaaa tagatttatc 240

cgtttgtgaa gtggctgctc gttcgatggt gcaaacggaa aaaaagaacc aaaaatccat 300

ttttccgaaa ccttcatagt tcctccgaaa tattcagagg tgaaaaagct cgagagagtt 360

cagacgcaca aaacatggct gaaccaggca cgaagttcca ctataccatc gaatatgatg 420

gatttgaaag cagatggtaa agcaaagaga gtgacggggt cattcaacga gtaatgggtt 480

gagcaagtga ttgcctagag gatgaaggag gtggtacttc tgtttgtcac tagcaggata 540

gaaaaaatta tcattatctc tcagaaacgt aatagaagcg ttttcacata gagctacggg 600

tttgcatata cttctgttag ttttgttcac acctgctagt tgttgcacat acgcaataag 660

atattttttg ataccggttg aagatggttc tcctcttatg tcggtcagct gtcgtcgctt720

taggtataat tctgtgctac gattacgtac agagtagata ttagagacca tggtataatt 780

caattgtata ttaaaactat gttgagaaat actggaagac agtaatcagt tgattaattc 840

gagaatcccc gtgtaccagc caagtagtgt gtaagtgtaa ttgcgtgtca cttctttttt 900

ttctctactg cttagtacct ttctttcttt tcctctcgta gggttgggaa tccagtattg 960

tgggctaatg aactgagtca cataatgtgg ttatgttcca atataggtac cacctttgtt 1020

caagatttag ttttctaatt gaatataaat acagaggtta tttcaaccta attgagatta 1080

aggagagact tattttacta tagtatatat ttatttataa ttacttattg ttactccaat 1140

ccccaagtag attagattta atcaatcaca cacaaacatc aaaacaacaa attaacaaaa 1200

<210>5

<211>1000

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>5

aaaaagacct agcggggccc ggaaaaatac ggtggtaaaa gtttttcgct atgattttcc 60

agagtttcac ttcagaagag ccagtggaaa ctcaaaacaa atatctccag attttttcgg 120

aataacatca tacattcaac acccaaatag aagatacgaa agcacgagat ataacagaaa 180

aggatgtgca gaacctctga ccttgcttac ctcgagagca ataaccaatt catatagctg 240

ataggtagtt acgtttacta cttaactact tttcccccaa ccaaatctga ttctaatccc 300

ccgcgtgaaa aaaaaaaaaa cacaaaagaa gagaacacag aataagatac aaccacagag 360

agtacgtgcg tacgtgcgtg catctcgcca cttccacttc gatttatctg ccccgcataa 420

actagagatt gaaaatgtgc tcttgtgcta gctcctccgc gacttctcgg taaaaccttg 480

cttctcctat tattcccgta gcgagcctgg aaaaatttca acaacaactt ttttttttca 540

gttcagcttc cacaaagttt cctcttccac aaagttcctt agaaacgaaa aacttcatat 600

agacggatcc catttgaatg tggaggcagg caggaattca gatgaatttg accttggaat 660

gtaaagcgca aaccactatt tggttccgac ttgacgttgc gagtggcagt ttctggatca 720

tttgcgtata taaatatata aatatgttgt tctatcgtag agttctttcc ttttcagttg 780

taggttttgt ccactgtgaa ttgttatacc atcatttctt aattacagac agccccaagt 840

tttaaagtat acaagtctca ttactactta ctactactac tactactgct gtgattaatt 900

gctattttac tagaaatact actagttata gcatcataag aagagtatta gagctaacgc 960

aaaagctaaa cttttagatt acattacata aatatcaaga 1000

<210>6

<211>1200

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>6

gggcctgccc tcagcactcg tagacacgag taacgtcttg agacctctcg tacagggaag 60

cgacatatcg ttcaatagac tatggaacaa agtgtacacc gcagcgatat ccttgcattt 120

gcaaaacgat tgaataagtg acgtcgatgc taaatcctgg ataagtacgc tggtatcgtg 180

taagcccatg agaacgacac gttcctcatc actagaagcc gaactgttgt cttcagtggg 240

gattggttcg acattttgcc aattgctgtc gatgtaccct ttcaaagcca tgtaccttaa 300

atcttcatcc ttggcaagta gattcatcgg gtgtgtttga agtaagaata tttgcttgtt 360

tttatggtat caaaggtata tgttgtagaa gacaatttcc ggtaatccaa ttgtctgtct 420

gctcagttta gcacatgtat agtacgttgc acatagtcta caatattcag cattcagcat 480

tcagtataca gcatatggct aaatgatcac aaatgtgatt gatgatttga cacgactaga 540

aaagagaacg aaaaagggaa attccatgtc acgtgcgttg gcacgtgaca tggaatatcg 600

aagaaagaaa aaaaaaacga tctcgtccta gtggaagccc agagtctggt ccccccggag 660

tcttcccaaa acaagaagct gacacatgtt gacacagaac accccacagc aaatgcacca 720

cgctacgtag atcaggaagc ttaactctag cgacctgtcg ctcgccccac agaacctcac 780

ccgagaacca cacattacac gccgccagct cccactatac tcatcttgct tcccttaagc 840

gttctcacga ttcgttcgct gcccttcttc aagagtcttc tgattctaat tctcattcga 900

aatcctctac agttaatgaa ttgcttgaca tgacattcat tgtctcatgg ttttggcttt 960

ttggcttttg tcttttaaag ctatatcaac tttacatata aatatacgtc aaaaggggat 1020

tcattaatta gaaaattctc tttttcaata gttgctattc attatcaatc tattcaactc 1080

aattggttat tattttcatc tttttgtcat cctaaaccat caacaatatt taaatatatc 1140

tgttgctaca ttaagagtta cttcagaaat aacaaaaaaa tcgatcaaga attaataaaa 1200

<210>7

<211>1200

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>7

acggtttcac gatgaaacca ctttagcgct gaagttggta agattcagaa ccagaactac 60

attcagtgct agaagtgtat gcagggcgtc tctggtttcg ctatgctact ggtggagtgg 120

atgtgaatgg cctcagtctc gcttttagag agagtcccac taagcagtcc aaagaaagct 180

cccactggaa caggggaaag gagcctgtcc aagcaaatgc cttctcataa atggtgccaa 240

agacccgcaa gcccaaagca attacccccc aaaaagaaat gatatagtgc aagatacgta 300

tatgaccatg acttgactag gtgaaacagt gcagaaacag ccgcacaaaa gcagccctaa 360

ccctcagagt cgattttact ctttcaggta ataaagcctc gacatcaatt ttagacagaa 420

gccaggctgg cctcgagatt atagccatag gcaagcaaga ggagagaagg ggaggccccc 480

catggggggc ctcccccccg ctgtcaaggt ttggcagaac ctagcttcat taggccacta 540

gcccagccta aaacgtcaac gggcaggagg aacactccca caagacggcg tagtattctc 600

gattcataac cattttctca atcgaattac acagaacaca ccgtacaaac ctctctatca 660

taactactta atagtcacac acgtactcgt ctaaatacac atcatcgtcc tacaagttca 720

tcaaagtgtt ggacagacaa ctataccagc atggatctct tgtatcggtt cttttctccc 780

gctctctcgc aataacaatg aacactgggt caatcatagc ctacacaggt gaacagagta 840

gcgtttatac agggtttata cggtgattcc tacggcaaaa atttttcatt tctaaaaaaa 900

aaaagaaaaa tttttctttc caacgctaga aggaaaagaa aaatctaatt aaattgattt 960

ggtgattttc tgagagttcc ctttttcata tatcgaattt tgaatataaa aggagatcga 1020

aaaaattttt ctattcaatc tgttttctgg ttttatttga tagttttttt gtgtattatt 1080

attatggatt agtactggtt tatatgggtt tttctgtata acttcttttt attttagttt 1140

gtttaatctt attttgagtt acattatagt tccctaactg caagagaagt aacattaaaa 1200

<210>8

<211>1000

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>8

gttcaacatt gttgaagacg tatcttggaa agatgatgtc atcaaagttt tcgaagacaa 60

gatggaaaga ctacccggtt cttactgcaa gatcggagat tcgatggtga gattccatac 120

agaaaatgca gaagaccgtg atcgtgtgca aggtgttatt ggcgaagcaa tgacacatat 180

caacaccttg ttttcagaca agggtatcca tgcctacgtt aataaaaaca ttgtgtttgt 240

gcaagaagtc ggattagcat tgaaaacgtt gcaattcttg gtgagctatt ataattctgt 300

tgatgacatt tcatgttcat ctcattctcc agtggcgcaa ccaacttctg ctgaaacgtc 360

cgcatccccc ctcctatctc caatatctcc aaagaacggt gctggtttct tccacgttgc 420

tgccaagagg gaaaatagag acccaattgg attcctaacg atcactgggt ccacgtctcc 480

cgttattgaa ccattgttcc aatacgtcaa tgaactcacg aaggaaggaa agcttcagta 540

cgggtactct gtagtacatg gtgatacgtc ttcaacatac gctaaagagc acattcaggg 600

tcttaacgag ctattttcga tgctacaaaa gttgtcttct cccaactcat cctaaaagag 660

atagaacaaa tctgatcctt cccataatat aaataaagag aatctgtata gtttacgata 720

atgtgataaa gatgctggtt taagatatgt gaggcataag caacgcatca gtcaccggta 780

cggacataaa tgcggtatcc tatgacatca cgtgatataa tcactctgga cgagttgaaa 840

aattttaggt ttcagaccaa acgcctacaa taagcaatga ctttaaagaa ctgatgagat 900

gaatagacta gattacttga ggttttaacg ttcatattgt tgttgtacta gtcttttagc 960

attctgcagt gatagaacta gaaaatccat aactgtaaag 1000

Claims

1. An in vitro protein synthesis system for improving the expression level of a foreign protein, wherein the synthesis system at least comprises a cell lysate, the cell lysate is from a genetic engineering cell, and a polynucleotide sequence constructed by inserting a strong promoter sequence in front of a nucleotide sequence for encoding eIF4E binding protein is integrated in a gene sequence of the genetic engineering cell; and no or additional eIF4E binding protein was added to the synthesis system.

2. The synthesis system according to claim 1, characterized in that: the strong promoter sequence is selected from one of GAPDH1, HXK1, PGK1, TEF1, TIF1, ADH1 and SED1 or the combination thereof.

3. The synthesis system according to claim 2, characterized in that: the strong promoter sequence is selected from one of pScGAPDH1, pKLHXK1, pKLPGK1, pScPGPK 1, pScSED1, pScADH1 and pKLGAPDH1 or the combination thereof.

4. The synthesis system according to any one of claims 1 to 3, characterized in that: the eIF4E binding protein is one or the combination of Eap1p, p20, 4E-BP1, 4E-BP2 and PHAS-I.

5. The synthesis system according to any one of claims 1 to 3, characterized in that: the cell is a eukaryotic cell.

6. The synthesis system according to claim 5, characterized in that: the eukaryotic cell is one of mammalian cell, plant cell, yeast cell, insect cell or any combination thereof.

7. The synthesis system according to claim 6, characterized in that: the yeast is selected from one of Saccharomyces cerevisiae and Kluyveromyces yeast or their combination.

8. The synthesis system according to claim 7, characterized in that: the yeast of the genus Kluyveromyces is selected from one of Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polybracteus, or any combination thereof.

9. A method for synthesizing a protein in vitro, comprising the steps of:

(i) providing a protein synthesis system, wherein said synthesis system is according to any one of claims 1-8; and

10. The in vitro protein synthesis method of claim 9, further comprising: (iii) isolating or detecting the foreign protein.