CN113584058B - Signal Peptide Related Sequence and Its Application in Protein Synthesis - Google Patents

Abstract

The present invention provides a signal peptide-related sequence and its application in protein synthesis. Specifically, the present invention provides a signal peptide and its coding sequence with the effect of improving protein expression. The coding sequence of the signal peptide and the coding sequence of the foreign protein can be The nucleic acid construct formed by the operation link can significantly improve the efficiency of exogenous protein synthesis, and simplify the expression and purification process of the target exogenous protein. At the same time, the present invention provides its corresponding vector or vector combination, genetically engineered cell, and kit, so that it can be applied in protein synthesis.

Description

Signal Peptide Related Sequence and Its Application in Protein Synthesis

technical field

The invention relates to the field of biotechnology, in particular to signal peptide-related sequences and their application in protein synthesis.

Background technique

Proteins are important molecules in cells and are involved in almost all functions of cells. Proteins vary in sequence and structure, determining their differences in function (1,2). In cells, proteins can act as enzymes to catalyze various biochemical reactions, act as signaling molecules to coordinate various activities of organisms, support biological forms, store energy, transport molecules, and enable organisms to move (2). In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating diseases such as cancer (2).

Signal peptides are short peptides located at the N-terminus of proteins that carry information about protein secretion, and they are widely distributed in all prokaryotes and eukaryotes (3,4). For the study of signal peptides, it is emphatically expressed in many scientific and industrial fields, including the production of recombinant proteins, disease diagnosis, immunity and many biological experimental techniques (4,5). Many studies have shown that signal peptides play a very important role in recombinant protein production (6,7). However, some functions of signal peptides in protein expression and formation of transmembrane structures remain obscure (4,8,9).

In addition to what is known about protein synthesis inside the cell, protein synthesis can also take place outside the cell. The in vitro protein synthesis system generally refers to the rapid and efficient translation of exogenous proteins by adding mRNA or DNA templates, RNA polymerase, amino acids, ATP and other components to the lysis system of bacteria, fungi, plant cells or animal cells (10, 11). At present, commercial in vitro protein expression systems that are frequently tested include E. coli system (E.coli extract, ECE), rabbit reticulocyte lysate (RRL), wheat germ (Wheat germ extract, WGE), insect (Insect cell extract, ICE) and human-derived systems (11,12). Compared with the traditional in vivo recombinant expression system, the in vitro cell-free protein synthesis system has many advantages, such as the ability to express special proteins that are toxic to cells or contain unnatural amino acids (such as D-amino acids), and can be directly expressed as PCR products. Simultaneously parallel synthesis of multiple proteins as a template for high-throughput drug screening and proteomics research (10-12).

Studies have shown that part of the signal peptide sequence can promote protein expression, but the DNA template used for in vitro synthesis usually does not have a signal peptide-related sequence (13). Therefore, in the in vitro protein synthesis system, a short polypeptide sequence with a length of less than 30 amino acids is generally inserted into the N-terminal of the target protein to improve the translation efficiency of the target protein, but the insertion of some short peptides will significantly affect the structure and function of the target protein (4 ,14).

Therefore, there is an urgent need in this field to provide a signal peptide-related sequence that can be applied to an in vitro protein expression system, which can significantly increase the yield of the target protein, reduce the cost of protein expression, and improve protein translation efficiency.

Contents of the invention

The purpose of the present invention is to provide a signal peptide-related sequence that can be applied in an in vitro protein expression system, can significantly increase the yield of the target protein, reduce the cost of protein expression, and improve protein translation efficiency.

The first aspect of the present invention provides a nucleic acid construct, the nucleic acid construct comprises a first nucleotide sequence encoding a signal peptide, which is operably linked to a second nucleotide sequence encoding a foreign protein, the first core The 3' end of the nucleotide sequence is upstream of the second nucleotide sequence, and the first nucleotide sequence is selected from the group consisting of:

(a) a nucleotide sequence encoding any of the following signal peptides: a signal peptide whose amino acid sequence is SEQ ID NO: 14-24;

(b) Any nucleotide sequence shown in SEQ ID NO: 1-13.

In another preferred example, the nucleic acid construct has a structure of formula I from 5' to 3':

Z1-Z2-Z3 (I)

In the formula,

Z1-Z3 are elements used to constitute the construct respectively;

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

Z1 is the coding sequence of the signal peptide;

Z2 is none or a linked sequence;

Z3 is the coding sequence of none or foreign protein;

Wherein, the coding sequence of the signal peptide is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.: 14-24;

(b) a polynucleotide whose sequence is any one of SEQ ID NO.: 1-13;

(c) The homology between the nucleotide sequence and any of the sequences shown in SEQ ID NO.: 1-13 is ≥75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) polynucleotides;

(d) truncating or adding 1-60 (preferably 1-30, more preferably 1- 10) polynucleotides of nucleotides;

(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).

In another preferred example, the nucleic acid construct has a structure of formula II from 5' to 3':

Z1-Z2-Z3 (II)

In the formula,

Z1-Z3 are elements used to constitute the construct respectively;

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

Z1 is the coding sequence of the signal peptide;

Z2 is a connecting sequence;

Z3 is the coding sequence of none or foreign protein;

Wherein, the coding sequence of the signal peptide is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.: 14-24;

(b) a polynucleotide whose sequence is any one of SEQ ID NO.: 1-13;

(c) The homology between the nucleotide sequence and any of the sequences shown in SEQ ID NO.: 1-13 is ≥75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) polynucleotides;

(d) truncating or adding 1-60 (preferably 1-30, more preferably 1- 10) polynucleotides of nucleotides;

(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).

In another preferred example, the nucleic acid construct has a structure of formula III from 5' to 3':

Z1-Z2-Z3 (III)

In the formula,

Z1-Z3 are elements used to constitute the construct respectively;

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

Z1 is the coding sequence of the signal peptide;

Z2 is a connecting sequence;

Z3 is the coding sequence of the foreign protein;

Wherein, the coding sequence of the signal peptide is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.: 14-24;

(b) a polynucleotide whose sequence is any one of SEQ ID NO.: 1-13;

(c) The homology between the nucleotide sequence and any of the sequences shown in SEQ ID NO.: 1-13 is ≥75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) polynucleotides;

(d) truncating or adding 1-60 (preferably 1-30, more preferably 1- 10) polynucleotides of nucleotides;

(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).

In another preferred example, the said operably linking is direct linking or linking through linking sequence.

In another preferred example, the linking sequence is the nucleotide sequence shown in SEQ ID NO:25.

In another preferred example, the amino acid sequence of the connecting sequence is shown in SEQ ID NO.:26.

In another preferred example, the coding sequence of the signal peptide is a codon-optimized coding sequence.

In another preferred example, the coding sequence of the signal peptide is shown in SEQ ID NO.: 1-13.

In another preferred example, the amino acid sequence of the signal peptide has the sequence shown in SEQ ID NO.: 14-24 or an active fragment thereof, or has an amino acid sequence ≥ 85% of the amino acid sequence shown in SEQ ID NO: 14-24 Homology, preferably ≥ 90% homology; More preferably ≥ 95% homology; Most preferably, ≥ 97% homology, such as 98% or more, 99% or more) and have the same identity as SEQ A polypeptide with the same activity as the sequence shown in ID NO.: 14-24.

In another preferred example, the coding sequence of the signal peptide is shown in SEQ ID NO.: 11-13.

In another preferred example, the coding sequence of the signal peptide is shown in SEQ ID NO.: 2-7.

In another preferred example, the coding sequence of the signal peptide is shown in SEQ ID NO.:1.

In another preferred example, the coding sequence of the signal peptide is shown in SEQ ID NO.: 8-10.

In another preferred example, the amino acid sequence of the signal peptide is shown in SEQ ID NO.: 22-24.

In another preferred example, the amino acid sequence of the signal peptide is shown in SEQ ID NO.: 15-20.

In another preferred example, the amino acid sequence of the signal peptide is shown in SEQ ID NO.:14.

In another preferred example, the amino acid sequence of the signal peptide is shown in SEQ ID NO.:21.

In another preferred example, the connecting sequence is a codon-optimized connecting sequence.

In another preferred example, the connecting sequence has a sequence that is not easy to form a secondary structure (such as AT-rich sequence, no hairpin structure, no G-quadruplex (G-quadruplex), etc.), and is not rich in rare codons .

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

(i) a polynucleotide whose sequence is as shown in SEQ ID NO.:25;

(ii) The homology between the nucleotide sequence and the sequence shown in SEQ ID NO.: 25 ≥ 75% (preferably ≥ 85%, more preferably ≥ 90% or ≥ 95% or ≥ 98% or ≥ 99% ) polynucleotide;

(iii) Truncating or adding 1-60 (preferably 1-30, more preferably 1-10) cores at the 5' end and/or 3' end of the polynucleotide shown in SEQ ID NO.:25 polynucleotides of nucleotides;

(iv) A polynucleotide complementary to the polynucleotide described in any one of (i)-(iii).

In another preferred example, the foreign protein is from prokaryotes or eukaryotes.

In another preferred example, the foreign protein is from animals, plants, or pathogens.

In another preferred example, the foreign protein is from mammals, preferably primates, rodents, including humans, mice, and rats.

In another preferred example, the exogenous protein is selected from the group consisting of luciferin, luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3 -Phosphate dehydrogenase, catalase, actin, variable regions of antibodies, luciferase mutations, alpha-amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor, Interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.

In another preferred example, the coding sequence of the foreign protein encodes a protein selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacid tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody or variable region thereof, luciferase mutant, or a combination thereof.

In another preferred example, the upstream of the 5' end of the nucleic acid construct further includes a promoter.

In another preferred example, the promoter includes a constitutive or inducible promoter.

In another preferred example, the promoter is selected from the group consisting of T7 promoter, T3 promoter, SP6 promoter, or a combination thereof.

In another preferred example, the nucleic acid construct further includes an enhancer element, an RBS ribosome binding sequence, a spacer, other related sequences for RNA transcription and translation, or a combination thereof.

In another preferred embodiment, the enhancer element includes an internal ribosome entry site element (IRES), a ribosome binding site element, a non-coding sequence, or a combination thereof.

In another preferred example, the source of the IRES element is one or more cells selected from the group consisting of prokaryotic cells and eukaryotic cells.

In another preferred example, the eukaryotic cells include higher eukaryotic cells.

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

In another preferred embodiment, the source of the IRES element is one or more cells selected from the group consisting of human (human), Chinese hamster ovary cell (Chinese hamster ovary cell, CHO), insect cell (insect), wheat germ (Wheatgerm cells), rabbit reticulocytes (Rabbit reticulocyte).

In another preferred embodiment, the IRES element is selected from the group consisting of ScGPR1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1, GAA, Omega, Omega10A, or a combination thereof.

The second aspect of the present invention provides a signal peptide whose amino acid sequence is encoded by the first nucleotide sequence in the first aspect.

In another preferred example, the amino acid sequence of the signal peptide is shown in any one of SEQ ID No.: 14-24.

The third aspect of the present invention provides a vector or a combination of vectors, which contains the nucleic acid construct of the first aspect of the present invention.

The fourth aspect of the present invention provides a genetically engineered cell, one or more sites of the genome of the genetically engineered cell is integrated with the nucleic acid construct in the first aspect of the present invention, or the genetically engineered cell contains the nucleic acid construct of the present invention The carrier or combination of carriers in the third aspect.

In another preferred example, the genetically engineered cells include prokaryotic cells and eukaryotic cells.

In another preferred example, the eukaryotic cells include higher eukaryotic cells.

In another preferred embodiment, the genetically engineered cells are selected from the group consisting of human cells (such as Hela cells), Chinese hamster ovary cells, insect cells, wheat germ cells, rabbit reticulocytes, yeast cells, or combinations thereof.

In another preferred example, the genetically engineered cells are yeast cells.

In another preferred embodiment, the yeast cell 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 marikus, Kluyveromyces dobui, or a combination thereof.

In the fifth aspect of the present invention, a kit is provided, and the reagents contained in the kit are selected from one or more of the following groups:

(a) the nucleic acid construct described in the first aspect of the present invention;

(b) the vector or combination of vectors described in the third aspect of the present invention; and

(c) the genetically engineered cell described in the fourth aspect of the present invention.

In another preferred example, the kit further includes (d) eukaryotic in vitro biosynthesis system (such as eukaryotic in vitro protein synthesis system).

In another preferred example, the eukaryotic in vitro biosynthesis system is selected from the group consisting of yeast in vitro biosynthesis system, Chinese hamster ovary cell in vitro biosynthesis system, insect cell in vitro biosynthesis system, Hela cell in vitro biosynthesis system, or its combination.

In another preferred example, the eukaryotic in vitro biosynthesis system includes a eukaryotic in vitro protein synthesis system.

In another preferred example, the eukaryotic in vitro protein synthesis system is selected from the group consisting of yeast in vitro protein synthesis system, Chinese hamster ovary cell in vitro protein synthesis system, insect cell in vitro protein synthesis system, Hela cell in vitro protein synthesis system, or its combination.

In another preferred embodiment, the yeast in vitro biosynthesis system (such as yeast in vitro protein synthesis system) is Kluyveromyces in vitro biosynthesis system (such as Kluyveromyces in vitro protein synthesis system), preferably Kluyveromyces lactis In vitro biosynthesis system (such as Kluyveromyces lactis in vitro protein synthesis system).

In another preferred example, the yeast in vitro biosynthesis system is Kluyveromyces in vitro biosynthesis system.

The sixth aspect of the present invention provides the nucleic acid construct described in the first aspect, the signal peptide described in the second aspect, the vector or combination of vectors described in the third aspect, the genetically engineered cell described in the fourth aspect, or the genetically engineered cell described in the fifth aspect. The application of the kit described above in the in vitro protein synthesis system.

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

(i) providing an in vitro biosynthesis system, said in vitro biosynthesis system containing the nucleic acid construct described in the first aspect of the present invention;

(ii) incubating the in vitro biosynthesis system of step (i) under appropriate conditions, and synthesizing the exogenous protein after a certain reaction time.

In another preferred embodiment, the in vitro protein synthesis method further includes (iii) optionally isolating or detecting the exogenous protein from the eukaryotic in vitro biosynthesis system.

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

In another preferred example, in the step (ii), the reaction time is 1-72h, preferably 2-24h.

In another preferred example, the in vitro biosynthesis system may be a yeast in vitro biosynthesis system (such as a yeast in vitro protein synthesis system).

In another preferred embodiment, the yeast in vitro biosynthesis system (such as yeast in vitro protein synthesis system) is Kluyveromyces in vitro biosynthesis system (such as Kluyveromyces in vitro protein synthesis system) (preferably Kluyveromyces lactis in vitro Biosynthesis system, such as Kluyveromyces lactis in vitro protein synthesis system).

In another preferred example, the foreign protein is from prokaryotes or eukaryotes.

In another preferred example, the foreign protein is from animals, plants, or pathogens.

In another preferred example, the foreign protein is from mammals, preferably primates, rodents, including humans, mice, and rats.

In another preferred example, the exogenous protein is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde- 3-phosphate dehydrogenase, catalase, actin, variable regions of antibodies, luciferase mutations, alpha-amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor , interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.

In another preferred example, the coding sequence of the foreign protein encodes a foreign protein selected from the group consisting of luciferin, luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, ammonia Acyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable regions of antibodies, luciferase mutants, alpha-amylase, enterocin A, gamma Hepatitis virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase , or a combination thereof.

The eighth aspect of the present invention provides an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.: 14-24;

(b) a polynucleotide whose sequence is any one of SEQ ID NO.: 1-13;

(c) The homology between the nucleotide sequence and any of the sequences shown in SEQ ID NO.: 1-13 is ≥75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) polynucleotides;

(d) truncating or adding 1-60 (preferably 1-30, more preferably 1- 10) polynucleotides of nucleotides;

(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).

In another preferred example, the polynucleotide is a nucleotide sequence encoding a signal peptide.

In another preferred example, the polynucleotide includes a DNA sequence.

The ninth aspect of the present invention provides a connection sequence, the connection sequence is selected from the following group:

(i) a polynucleotide whose sequence is as shown in SEQ ID NO.:25;

(ii) The homology between the nucleotide sequence and the sequence shown in SEQ ID NO.: 25 ≥ 75% (preferably ≥ 85%, more preferably ≥ 90% or ≥ 95% or ≥ 98% or ≥ 99% ) polynucleotide;

(iii) Truncating or adding 1-60 (preferably 1-30, more preferably 1-10) cores at the 5' end and/or 3' end of the polynucleotide shown in SEQ ID NO.:25 polynucleotides of nucleotides;

(iv) A polynucleotide complementary to the polynucleotide described in any one of (i)-(iii).

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the basic biological process from DNA to protein.

Fig. 2 shows the relative fluorescence unit values (Relative Fluorescence Units, RFUs) of enhanced green fluorescent protein (Enhanced green fluorescent protein, eGFP) synthesized initially in an in vitro protein synthesis system by 13 signal peptide-related sequences of the present invention.

Detailed ways

After extensive and in-depth research, through a large number of screening and exploration, the inventors first discovered a novel signal peptide for improving the protein translation efficiency of an in vitro protein synthesis system and a nucleic acid construct comprising the signal peptide coding sequence. The nucleic acid of the present invention The construct includes a first nucleotide sequence encoding a signal peptide (including codon-optimized or non-optimized signal peptide encoding sequences) operably linked to a second nucleotide sequence encoding an exogenous protein. Experiments have shown that when the nucleic acid construct or signal peptide sequence of the present invention is applied in an in vitro protein synthesis system (such as a yeast in vitro protein synthesis system), the signal intensity of the synthesized foreign protein is significantly improved compared with the control group (p<0.05) . The present invention also simplifies the expression and purification methods of foreign proteins.

protein synthesis system

Protein synthesis refers to the process in which organisms synthesize proteins according to the genetic information on messenger ribonucleic acid (mRNA) transcribed from deoxyribonucleic acid (DNA), as shown in Figure 1. Protein biosynthesis is also called translation (Translation), which is the process of changing the sequence of bases in mRNA molecules to the sequence of amino acids in proteins or polypeptide chains. This is the second step in gene expression, the final stage in which the gene product protein is produced. Different tissue cells have different physiological functions because they express different genes and produce proteins with special functions. There are at least 200 kinds of components involved in protein biosynthesis, and the main body is composed of mRNA, tRNA, ribonucleoprotein and Associated enzymes and protein factors together.

In vitro protein synthesis system generally refers to adding mRNA or DNA template, RNA polymerase, amino acid, ATP and other components to the lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of foreign proteins. At present, commercial in vitro protein expression systems that are frequently tested include E. coli system (E.coli extract, ECE), rabbit reticulocyte lysate (RRL), wheat germ (Wheat germ extract, WGE), insect (Insect cell extract,ICE) and human source system.

Yeast has the advantages of simple culture, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms expressing complex eukaryotic proteins and membrane proteins, and yeast can also be used as raw materials for preparing in vitro translation systems.

Kluyveromyces is an ascospore yeast, among which Kluyveromyces marxianus and Kluyveromyces lactis are widely used industrially. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as super secretion ability, better large-scale fermentation characteristics, food safety level, and the ability to modify proteins at the same time.

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

In the present invention, the in vitro protein synthesis system includes: yeast cell extract and an optional solvent, and the solvent is water or an aqueous solvent.

In a particularly preferred embodiment, the in vitro protein synthesis system provided by the present invention also includes: 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, nucleoside triphosphate mixture, amino acid mixture, phosphocreatine, Dithiothreitol (DTT), Creatine Phosphokinase, RNase Inhibitor, Luciferin, 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 T7 RNA polymerase.

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

In the present invention, the yeast cell extract does not contain intact cells, and a typical yeast cell extract includes ribosomes for protein translation, transfer RNA, aminoacyl tRNA synthetase, initiation factors required for protein synthesis and elongation factor and termination release factor. In addition, yeast extract also contains some other proteins, especially soluble proteins, derived from the cytoplasm of yeast cells.

In the present invention, the protein content of the yeast cell extract is 10-100 mg/mL, preferably 20-80 mg/mL. The method for determining the protein content is a Coomassie brilliant blue method.

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

(i) providing yeast cells;

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

(iii) performing cell disruption treatment on the washed yeast cells, thereby obtaining crude yeast extract;

(iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

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

In the present invention, the centrifugation conditions are not particularly limited, and the centrifugation conditions are 5000-100000g, preferably 8000-30000g.

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

In the present invention, the temperature of the centrifugation is not particularly limited. Preferably, the centrifugation is performed at 1-10°C, more preferably at 2-6°C.

In the present invention, the washing treatment method is not particularly limited, and the preferred washing treatment method is to use a washing solution at a pH of 7-8 (preferably, 7.4) for treatment, and the washing solution is not particularly limited. Typically the washing solution is selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or combinations thereof.

In the present invention, the method of cell disruption treatment is not particularly limited, and preferred cell disruption treatment includes high-pressure disruption, freeze-thaw (such as 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 uridine nucleoside triphosphate. In the present invention, the concentration of various mononucleotides is not particularly limited, usually the concentration of each mononucleotide is 0.5-5 mM, preferably 1.0-2.0 mM.

The amino acid mixture in the in vitro protein synthesis system may include natural or unnatural amino acids, and may include D-type or L-type 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, 0.08mM.

In a preferred example, the in vitro protein synthesis system further contains polyethylene glycol (PEG) or its analogues. The concentration of polyethylene glycol or its analogs is not particularly limited, usually, the concentration (w/v) of polyethylene glycol or its analogs is 0.1-8%, preferably, 0.5-4%, more preferably, 1-2%, based on the total weight of the protein synthesis system. Representative examples of PEGs include, but are not limited to: PEG3000, PEG8000, PEG6000 and PEG3350. It should be understood that the system of the present invention may also include other polyethylene glycols of various molecular weights (such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).

In a preferred example, the in vitro protein synthesis system also contains sucrose. The concentration of sucrose is not particularly limited, usually, the concentration of sucrose is 0.03-40wt%, preferably 0.08-10wt%, more preferably 0.1-5wt%, based on the total weight of the protein synthesis system.

A particularly preferred system for in vitro protein synthesis contains, in addition to yeast extract, the following components: 22 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM Magnesium acetate, 1.5-4mM nucleoside triphosphate mixture, 0.08-0.24mM amino acid mixture, 25mM phosphocreatine, 1.7mM dithiothreitol, 0.27mg/mL phosphocreatine kinase, 1%-4% polyethylene glycol Alcohol, 0.5%-2% sucrose, 8-20ng/μL firefly luciferase DNA, 0.027-0.054mg/mL T7 RNA polymerase.

Coding sequence of foreign protein (foreign DNA)

As used herein, the terms "coding sequence of foreign protein" and "foreign DNA" are used interchangeably, and both refer to foreign DNA molecules used to direct protein synthesis. Typically, the DNA molecules are linear or circular. Said DNA molecules contain sequences encoding foreign proteins.

In the present invention, examples of the sequence encoding the foreign protein include (but not limited to): genome sequence, cDNA sequence. The sequence encoding the foreign protein also contains a promoter sequence, a 5' non-translation sequence, and a 3' non-translation sequence.

In the present invention, the selection of the exogenous DNA is not particularly limited. Usually, the exogenous DNA encodes a protein selected from the following group: luciferin, luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein Protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody or variable region thereof, luciferase mutant, or a combination thereof.

The exogenous DNA can also encode a protein selected from the group consisting of alpha-amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, Serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.

In a preferred embodiment, the exogenous DNA encodes a protein selected from the group consisting of green fluorescent protein (enhanced GFP, eGFP), yellow fluorescent protein (YFP), Escherichia coli β-galactosidase (β-galactosidase, LacZ), human lysine-tRNA synthetase (Lysine-tRNA synthetase), human leucine-tRNA synthetase (Leucine-tRNA synthetase), Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphatedehydrogenase ), mouse catalase (Catalase), or a combination thereof.

nucleic acid construct

The first aspect of the present invention provides a nucleic acid construct, which comprises a first nucleotide sequence encoding a signal peptide, which is operably linked to a second nucleotide sequence encoding a foreign protein, the first The 3' end of a nucleotide sequence is upstream of the second nucleotide sequence, and the first nucleotide sequence is selected from the group consisting of:

(a) a nucleotide sequence encoding any of the following signal peptides: a signal peptide whose amino acid sequence is SEQ ID NO: 14-24;

(b) Any nucleotide sequence shown in SEQ ID NO: 1-13.

The term "operably linked" refers to the functional spatial arrangement of two or more nucleotide regions or nucleotide sequences. For example: the nucleotide sequence encoding the signal peptide is placed at a specific position relative to the nucleotide sequence of the foreign protein, so that the effect of improving the expression of the foreign protein can be obtained. Said operably linking is direct linking or linking through linking sequence.

In a preferred embodiment, the nucleic acid construct of the present invention has a structure of formula I from 5' to 3':

Z1-Z2-Z3 (I)

In the formula,

Z1-Z3 are elements used to constitute the construct respectively;

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

Z1 is the coding sequence of the signal peptide;

Z2 is none or a linked sequence;

Z3 is the coding sequence of none or foreign protein;

Wherein, the coding sequence of the signal peptide is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.: 14-24;

(b) a polynucleotide whose sequence is any one of SEQ ID NO.: 1-13;

(c) The homology between the nucleotide sequence and any of the sequences shown in SEQ ID NO.: 1-13 is ≥75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) polynucleotides;

(d) truncating or adding 1-60 (preferably 1-30, more preferably 1- 10) polynucleotides of nucleotides;

(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).

In a preferred embodiment, the nucleic acid construct of the present invention has a formula II structure from 5' to 3':

Z1-Z2-Z3 (II)

In the formula,

Z1-Z3 are elements used to constitute the construct respectively;

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

Z1 is the coding sequence of the signal peptide;

Z2 is a connecting sequence;

Z3 is the coding sequence of none or foreign protein;

Wherein, the coding sequence of the signal peptide is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.: 14-24;

(b) a polynucleotide whose sequence is any one of SEQ ID NO.: 1-13;

(c) The homology between the nucleotide sequence and any of the sequences shown in SEQ ID NO.: 1-13 is ≥75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) polynucleotides;

(d) truncating or adding 1-60 (preferably 1-30, more preferably 1- 10) polynucleotides of nucleotides;

(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).

In a preferred embodiment, the nucleic acid construct of the present invention has a structure of formula III from 5' to 3':

Z1-Z2-Z3 (III)

In the formula,

Z1-Z3 are elements used to constitute the construct respectively;

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

Z1 is the coding sequence of the signal peptide;

Z2 is a connecting sequence;

Z3 is the coding sequence of the foreign protein;

Wherein, the coding sequence of the signal peptide is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.: 14-24;

(b) a polynucleotide whose sequence is any one of SEQ ID NO.: 1-13;

(c) The homology between the nucleotide sequence and any of the sequences shown in SEQ ID NO.: 1-13 is ≥75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) polynucleotides;

(d) truncating or adding 1-60 (preferably 1-30, more preferably 1- 10) polynucleotides of nucleotides;

(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).

In a preferred embodiment, the amino acid sequence of the signal peptide of the present invention has the sequence shown in SEQ ID NO.: 14-24 or an active fragment thereof, or has an amino acid sequence ≥ 85% different from the amino acid sequence shown in SEQ ID NO: 14-24 % homology, preferably ≥ 90% homology; more preferably ≥ 95% homology; most preferably, ≥ 97% homology, such as 98% or more, 99% or more) and have A polypeptide having the same activity as the sequence shown in SEQ ID NO.: 14-24.

In the present invention, the selection of the coding sequence of the foreign protein is not particularly limited. Usually, the coding sequence of the foreign protein encodes a protein selected from the group consisting of luciferin, luciferase (such as firefly luciferase), Green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody or variable region thereof, luciferase mutant, or combinations thereof .

The coding sequence of the foreign protein may also encode a protein selected from the group consisting of alpha-amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme Enzyme, serum albumin, single chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.

In addition, the nucleic acid construct of the present invention can be linear or circular. The nucleic acid construct of the present invention can be single-stranded or double-stranded. The nucleic acid construct of the present invention can be DNA, RNA, or DNA/RNA hybrid.

The preferred signal peptide sequence of the present invention and the nucleotide sequence encoding the signal peptide are shown in Table 1.

In another preferred example, the construct further comprises elements selected from the following group or combinations thereof: promoters, terminators, poly(A) elements, transport elements, gene targeting elements, screening marker genes, enhancers , resistance gene, transposase coding gene.

A variety of selectable marker genes can be used in the present invention, including but not limited to: auxotrophic markers, resistance markers, reporter gene markers. The use of selectable markers allows for the selection of recombinant cells (recombinants), allowing recipient cells to be distinguished from non-transformed cells. The auxotrophic marker is the complementation of the transferred marker gene with the mutant gene of the recipient cell, so that the recipient cell expresses wild-type growth. Resistance markers refer to the transfer of resistance genes into recipient cells, and the transferred genes make recipient cells resistant to drugs at a certain drug concentration. As a preferred mode of the present invention, a resistance marker is used to realize convenient screening of recombinant cells.

In the present invention, the application of the nucleic acid construct of the present invention in an in vitro protein synthesis system can significantly improve the translation efficiency of foreign proteins. In a preferred embodiment, applying the nucleic acid construct of the present invention in the yeast in vitro protein synthesis system of the present invention can significantly improve the efficiency of protein translation.

carrier

The present invention also provides a vector or combination of vectors, said vector containing the nucleic acid construct of the present invention. Preferably, the vector is selected from bacterial plasmids, phages, yeast plasmids, animal cell vectors, and shuttle vectors. Furthermore, the vector may be a transposon vector. Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host.

Those skilled in the art can use well-known methods to construct an expression vector containing the promoter and/or target gene sequence of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like.

genetically engineered cells

The present invention also provides a genetically engineered cell, the genetically engineered cell contains the construct or vector or vector combination, or the genetically engineered cell chromosome is integrated with the construct or vector. In another preferred example, the genetically engineered cell further includes a vector containing a transposase gene or a transposase gene integrated on its chromosome.

Preferably, the genetically engineered cells are eukaryotic cells.

In another preferred embodiment, the eukaryotic cells include, but are not limited to: yeast cells (preferably, Kluyveromyces cells, more preferably Kluyveromyces cells).

The construct or vector of the present invention can be used to transform appropriate genetically engineered cells. Genetically engineered cells can be prokaryotic cells, such as Escherichia coli, Streptomyces, and Agrobacterium; or lower eukaryotic cells, such as yeast cells; or higher animal cells, such as insect cells. Those of ordinary skill in the art know how to select appropriate vectors and genetically engineered cells. Transformation of genetically engineered cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism (such as Escherichia coli), it can be treated with CaCl ₂ or electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Transformation of plants can also use methods such as Agrobacterium transformation or gene gun transformation, such as leaf disk method, immature embryo transformation method, flower bud soaking method and the like.

In addition, the genetically engineered cells of the present invention can be used to produce or provide the nucleic acid constructs of the present invention.

In vitro high-throughput protein synthesis method

The signal peptide and the construct containing the signal peptide coding sequence of the present invention are particularly suitable for significantly improving the synthesis efficiency or yield of foreign protein in an in vitro biosynthesis system.

Correspondingly, the present invention also provides an in vitro high-throughput protein synthesis method, comprising the steps of:

(i) in the presence of an in vitro protein synthesis system, providing the nucleic acid construct described in the first aspect of the present invention;

(ii) incubating the in vitro protein synthesis system of step (i) for a period of time T1 under suitable conditions, thereby synthesizing the foreign protein.

In another preferred example, the method further includes: (iii) optionally isolating or detecting the exogenous protein from the in vitro protein synthesis system.

The main advantages of the present invention include:

(1) The present invention finds for the first time that the signal peptide-related sequence and the coding sequence of the foreign protein are used as nucleic acid constructs in the in vitro protein synthesis system of the present invention, which can be used to improve the translation efficiency of the target protein and enable expression and purification.

(2) After the translation start codon, the signal peptide-related sequence of the present invention can affect the folding of mRNA, thereby changing the translation efficiency of the target protein.

(3) Compared with other cells, Kluyveromyces lactis can be used in the production of proteins in the food and pharmaceutical fields due to its safety and high efficiency, plus the advantages of in vitro protein synthesis systems, such as adapting to high-throughput proteins Synthesis screening, synthesis of toxic proteins, short time and low cost, so the in vitro protein synthesis system derived from Kluyveromyces lactis cells can also be widely used in related fields.

(4) The signal peptide-related sequence provided by the present invention can not only improve the translation efficiency of the target exogenous protein, but more importantly, can increase the ability of the yeast in vitro protein synthesis system (such as the Kluyveromyces lactis in vitro protein synthesis system) to synthesize different proteins. possibility.

(5) The present invention firstly develops a novel signal peptide for improving the protein translation efficiency of the in vitro protein synthesis system and a nucleic acid construct comprising the signal peptide coding sequence. The first nucleotide sequence of the coding signal peptide (such as the coding sequence of the codon-optimized signal peptide) that the dinucleotide sequence is operably linked, the nucleic acid construct of the present invention can significantly improve the expression of the foreign protein that is synthesized .

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, generally according to conventional conditions, such as people such as Sambrook, molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Material

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

The exogenous protein in the embodiment is eGFP as an example.

Example 1 Determination of eukaryotic cell signal peptide-related sequences

1.1 Source and determination of signal peptide-related sequences: Randomly intercept the DNA sequence corresponding to the N-terminus of the constructed foreign protein, and screen the sequence or element that significantly improves the expression of the foreign protein through experiments.

Specifically, select and synthesize 30 nucleotide fragments with a base length of 36, 54 or other lengths, and modify some bases by means of staggered replacement of synonymous codons to improve the success rate of plasmid construction, wherein Including (but not limited to): reducing the annealing temperature of the sequence in order to reduce the GC content in the sequence related to the signal peptide; or using preferred codons. Dozens of plasmids were constructed. After analysis and screening, 30 of them were tested for their effects on foreign protein expression. The results showed that compared with the control, 13 signal peptide-related sequences were experimentally verified to have the effect of improving protein expression. , the amino acid sequence of the corresponding signal peptide and the nucleotide sequence information encoding the signal peptide are listed in Table 1. Other sequence information that has no effect or insignificant effect on improving protein expression is not listed.

Table 1 Plasmids and related nucleic acid sequences

Embodiment 2 Containing the construction of the in vitro protein synthesis system plasmid of signal peptide related sequence

Plasmid construction: Use 1 pair of primers to amplify the selected 30 signal peptide-related sequences and connecting sequences (the connecting sequences contain TEV restriction sites), and use its original plasmid backbone containing the target protein (eGFP as an example) The corresponding reverse primers were used for amplification. After the amplification is completed, 30 signal peptide-related sequences + connecting sequence fragments are inserted into the N-terminal of the target protein. In the final constructed plasmid, 30 signal peptide-related sequences + linker sequence nucleic acid sequences were inserted between the ATG start codon of the pD2P-eGFP plasmid and eGFP. The names of 13 plasmids are: pD2P-1.0SP-(001-013) (see Table 1).

The specific construction process is as follows:

Use 2 pairs of primers to carry out PCR amplification respectively, and take 10 μL of the correctly identified amplified products and mix them; add 0.5 μL DpnI to the 10 μL amplified products, and incubate at 37°C for 6 hours; add 4 μL of DpnI-treated products to 50 μL DH5α competent In cells, place on ice for 30 minutes, heat shock at 42°C for 45s, place on ice for 3 minutes, add 200 μL LB liquid medium and shake at 37°C for 4 hours, spread on LB solid medium containing Amp antibiotics and culture overnight; pick 6 After a single clone is expanded and cultured, the plasmid is extracted and stored after sequencing to confirm the correctness.

Example 3 Application of Signal Peptide Related Sequences in In Vitro Protein Synthesis System

3.1 Using PCR, and using primers pD2P_F: CGCGAAATTAATACGACTCACTATAGG (SEQ ID No.: 27) and pD2P_R: TCCGGATATAGTTCCCTCCTTTCAG (SEQ ID NO.: 28), all plasmids are located between the T7 transcription start sequence and the termination sequence. Sequence fragments and pD2P-eGFP fragments were amplified.

Purify and enrich the amplified and correctly sequenced DNA fragments identified by ethanol precipitation: add 1/10 volume of 3M sodium acetate (pH5.2) to the PCR product, and then add 2.5-3 times more volume (this volume is the volume after adding sodium acetate) of 95% ethanol, placed on ice and incubated for 15 min; centrifuged at a speed higher than 14000g for 30 min at room temperature, discarded the supernatant; washed with 70% ethanol, and then Then centrifuge for 15min, discard the supernatant, dissolve the precipitate with ultrapure water, and measure the DNA concentration.

3.2 According to the instructions, add the purified DNA fragments to the self-made in vitro protein synthesis system. And the above reaction system was placed in an environment of 22-30° C., and incubated for about 2-6 hours. Immediately after the reaction was completed, it was placed in an Envision2120 multifunctional microplate reader (Perkin Elmer) for reading to detect the strength of the eGFP signal, and the relative fluorescence unit value (Relative Fluorescence Unit, RFU) was used as the activity unit.

PC (Positive Control) is an experimental group in which only linker sequences are added to the N-terminus of enhanced green fluorescent protein, and NC (Negative Control) is an experimental group in which no nucleic acid constructs are added. 1 μl, 2 μl, and 3 μl are the amounts of DNA templates added to the in vitro protein synthesis system, respectively, and the total reaction system volume of all reactions is 30 μl.

Experimental results

1. Construction of plasmids for in vitro protein synthesis system

After many attempts, 30 in vitro protein synthesis system plasmids containing signal peptide-related sequences were successfully constructed.

2. Application of signal peptide-related sequences in in vitro protein synthesis system

As shown in Figure 2, the 13 signal peptide-related sequences caused a significant increase in the RFU value of eGFP in the in vitro protein synthesis system (the RFU value reached more than 1500 after 3 hours of reaction), up to 2900. In particular, pD2P-1.0SP-012 (adding 1 μl of DNA template, the RFU value reached 2900 after 3 hours of reaction), compared with the control PC (RFU value 800) without inserting the signal peptide related sequence, the relative fluorescence unit value increased by 2.6 times.

For the other 17 unlisted signal peptide sequences, their relative fluorescence unit values did not change or did not change significantly compared with PC, mostly 800-830, such as 823 for pD2P-1.0SP-019, 823 for pD2P- 1.0SP-027 is 816.

The results of the present invention show that the signal peptide-related sequence at the N-terminal of the target protein can significantly increase the yield of the target protein, and greatly improve the expression and purification effect of the target protein. It improves the translation efficiency of the target protein and increases the selectivity of the protein expression and purification method of the in vitro synthesis system, which greatly enhances the usability of the in vitro protein synthesis system.

Moreover, the research of the present invention also found that 5'-UTR, strong promoters (such as T7 promoter, T3 promoter, SP6 promoter), different IRES elements (such as KLNCE102) and different signal peptide related sequences, 3 Combinations such as '-UTR can also further improve the translation efficiency of the target protein.

Comparative example (PC and NC)

PC (Positive Control) is the experimental group in which only the junction sequence is added to the N-terminus of the enhanced green fluorescent protein. When 1 μl of DNA template is added, the RFU value of the exogenous protein is 800, and the total volume of the reaction system is 30 μl.

NC (Negative Control) is the experimental group without adding any nucleic acid constructs, the RFU value of the exogenous protein is 20, and the total volume of the reaction system is 30 μl.

Among them, 1 μl, 2 μl, and 3 μl in Figure 2 represent the amount of DNA template added to the in vitro protein synthesis system, the total reaction system volume of all reactions is 30 μl, and the reaction time is 3 hours.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

references:

1. Fromm HJ, Hargrove M. Essentials of Biochemistry. 2012.

2. Garcia RA, Riley MR. Applied biochemistry and biotechnology. Humana Press, ; 1981.263-264p.

3. Martoglio B. Intramembrane proteolysis and post-targeting functions of signal peptides. Biochem Soc Trans. 2003; 31(6):1243–7.

4. Owji H, Nezafat N, Negahdaripour M, Hajiebrahimi A, Ghasemi Y. A Comprehensive Review of Signal Peptides: Structure, Roles, and Applications. EurJ Cell Biol. 2018.

5. Liu H, Wu R, Yuan L, Tian G, Huang X, Wen Y, et al. Introducing acleavable signal peptide enhances the packaging efficiency of lentiviral vectors pseudotyped with Japanese encephalitis virus envelope proteins. VirusRes. 2017; 229:9–16 .

6. Cui Y, Meng Y, Zhang J, Cheng B, Yin H, Gao C, et al. Efficient secretory expression of recombinant proteins in Escherichia coli with a novelactinomycete signal peptide. Protein Expr Purif. 2017; 129:69–74.

7. Ling HL, Rahmat Z, Murad AMA, Mahadi NM, Illias RM. Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli. Process Biochem. 2017; 61:47–55.

8. Zhang S, Corin K.18-Peptide surfactants in membrane protein purification and stabilization A2-Koutsopoulos, Sotirios BT-Peptide Applications in Biomedicine, Biotechnology and Bioengineering. In Woodhead Publishing; 2018.p.485–512.

9. Stone TA, Deber CM. Therapeutic design of peptide modulators of protein-protein interactions in membranes. Biochim Biophys Acta-Biomembr. 2017; 1859(4):577–85.

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

11. 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.

12. Lu Y. Cell-free synthetic biology: Engineering in an openworld. Synth Syst Biotechnol. 2017; 2(1):23–7.

13. Kralicek A V., Radjainia M, Mohamad Ali NAB, Carraher C, Newcomb RD, Mitra AK. A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags. Protein Expr Purif. 2011; 80(1): 117–24.

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sequence listing

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Claims (14)

1. A nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide, which is operably linked to a second nucleotide sequence encoding a foreign protein, 3 of the first nucleotide sequence The 'end is upstream of the second nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence encoding the following signal peptide: a signal peptide whose amino acid sequence is SGGQIFVKTLTG. 2. The nucleic acid construct according to claim 1, characterized in that: said operably linked is directly linked or linked via a linking sequence. 3. The nucleic acid construct according to claim 2, characterized in that: the linking sequence is the nucleotide sequence shown in SEQ ID NO.:25. 4. A signal peptide, characterized in that its amino acid sequence is encoded by the first nucleotide sequence in claim 1. 5. A vector or a combination of vectors, characterized in that the vector or combination of vectors contains the nucleic acid construct according to any one of claims 1-3. 6. A genetically engineered cell, characterized in that, one or more sites of the genome of the genetically engineered cell are integrated with the nucleic acid construct described in any one of claims 1-3, or the genetically engineered cell contains The carrier or combination of carriers according to claim 5; The genetically engineered cells are selected from prokaryotic cells, Chinese hamster ovary cells, insect cells, rabbit reticulocytes, yeast cells, or combinations thereof. 7. A kit, characterized in that the reagents contained in the kit are selected from one or more of the following groups: (a) the nucleic acid construct described in any one of claims 1-3; (b) the vector or combination of vectors of claim 5; and (c) the genetically engineered cell according to claim 6. 8. The kit according to claim 7, further comprising (d) an in vitro biosynthesis system. 9. The kit according to claim 8, characterized in that: the in vitro biosynthesis system is selected from the group consisting of yeast in vitro biosynthesis system, Chinese hamster ovary cell in vitro biosynthesis system, insect cell in vitro biosynthesis system, Hela A cell in vitro biosynthesis system, or a combination thereof. 10. The kit according to claim 9, characterized in that: the yeast in vitro biosynthesis system is Kluyveromyces in vitro biosynthesis system. 11. A nucleic acid construct as claimed in any one of claims 1-3, the signal peptide as claimed in claim 4, the vector or combination of vectors as claimed in claim 5, the genetically engineered cell as claimed in claim 6 or the Application of any one of the kits in requirements 7-9 in an in vitro protein synthesis system. 12. A method for in vitro protein synthesis, characterized in that it comprises the following steps: (i) providing an in vitro biosynthesis system, said in vitro biosynthesis system containing the nucleic acid construct described in any one of claims 1-3; (ii) incubating the in vitro biosynthesis system in step (i) for a certain period of time under suitable conditions, thereby synthesizing the foreign protein. 13. The in vitro protein synthesis method according to claim 12, characterized in that the suitable conditions are that the reaction temperature is 20-37°C and the reaction time is 1-72h. 14. The method for in vitro protein synthesis according to claim 12, further comprising: (iii) isolating or detecting the exogenous protein.

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