CN110408635B

CN110408635B - Application of nucleic acid construct containing streptavidin element in protein expression and purification

Info

Publication number: CN110408635B
Application number: CN201810402739.3A
Authority: CN
Inventors: 郭敏; 徐开; 章小铃; 姜灵轩; 王海鹏; 杨宁; 于雪
Original assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Current assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Priority date: 2018-04-28
Filing date: 2018-04-28
Publication date: 2021-07-30
Anticipated expiration: 2038-04-28
Also published as: CN110408635A

Abstract

The invention provides application of a nucleic acid construct containing a streptavidin element in protein expression and purification, and particularly provides a nucleic acid construct, wherein the nucleic acid construct has a structure from 5 'to 3' and is shown as a formula I: Z1-Z2-Z3 (I); wherein Z1, Z2 and Z3 are elements for constituting the construct, respectively; each "-" is independently a bond or a nucleotide linking sequence; z1 is the coding sequence of the tag protein; z2 is a linker sequence; z3 is the coding sequence of no or foreign protein, and the application of the nucleic acid construct in the protein synthesizing system (especially in vitro protein synthesizing system) can complete the expression and purification of foreign protein and raise the RFU value of the synthesized foreign protein.

Description

Application of nucleic acid construct containing streptavidin element in protein expression and purification

Technical Field

The invention relates to the field of biotechnology, in particular to application of a nucleic acid construct containing a streptavidin element in protein expression and purification.

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 proteins determines their function (1). In cells, proteins can catalyze various biochemical reactions as enzymes, can coordinate various activities of organisms as signaling molecules, can support biomorphic, store energy, transport molecules, and mobilize organisms (1). In the biomedical field, protein antibodies are important means for treating diseases such as cancer as targeted drugs (1, 2).

The yeast expression system, as a latter foreign protein expression system, has the advantages of both prokaryotic and eukaryotic expression systems, is increasingly widely applied in the field of genetic engineering, can express proteins at high levels by applying the system, and has a post-translational modification function, so the yeast expression system is recognized as a powerful tool (3,4) for expressing large-scale proteins.

Some Streptavidin (Streptavidin) and Avidin (Avidin) tags are found in cells from yeast to humans, but studies on intracellular Streptavidin and Avidin tags are not sufficient, mainly because intracellular Streptavidin tags affect the efficiency of initiation of protein translation and are prone to nonspecific binding during purification (4, 5). Unlike the simpler expression of proteins in viruses, the endogenous expression of proteins in different cells does not have sequence and structural commonality, and high throughput screening based on binding of biotin is often difficult (5, 6).

In addition to the above knowledge of intracellular protein synthesis, protein synthesis can also be carried out extracellularly. The in vitro protein synthesis system is generally characterized in that mRNA or DNA template, RNA polymerase, amino acid, ATP and other components are added into a lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of foreign proteins (7, 8). Currently, commercial in vitro protein expression systems that are frequently tested include the e.coli system (ECE), Rabbit Reticulocyte Lysate (RRL), Wheat Germ (WGE), Insect Cell Extract (ICE) and human-derived system (7). Compared with the traditional in vivo recombinant expression system, the in vitro cell-free protein synthesis system has multiple advantages, such as the capability of expressing special proteins which have toxic action on cells or contain unnatural amino acids (such as D-amino acids), the capability of simultaneously synthesizing multiple proteins in parallel by directly taking PCR products as templates, and the development of high-throughput drug screening and proteomics research (7, 9).

However, DNA templates used for in vitro synthesis do not typically have Streptavidin elements, and designing different tags for different proteins and purification methods is relatively time and cost intensive (10). The use of eukaryotic Streptavidin elements to initiate protein expression and purification in vitro is currently less than desirable.

Therefore, there is an urgent need in the art to develop a nucleic acid construct containing streptavidin that can be used for protein expression and purification in eukaryotic cells, and the nucleic acid construct can be applied to an in vitro protein expression system, so that the protein can be more conveniently purified, the purification efficiency is improved, and the cost is reduced.

Disclosure of Invention

The invention aims to provide a nucleic acid construct containing streptavidin, which can be used for protein expression and purification in eukaryotic cells.

In a first aspect, the invention provides a nucleic acid construct having a structure of formula I from 5 'to 3':

Z1-Z2-Z3 (I)

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

z1, Z2, Z3 are the elements used to construct the construct, respectively;

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

z1 is the coding sequence of the tag protein;

z2 is a linker sequence;

z3 is the coding sequence of no or foreign protein.

In another preferred embodiment, the tag protein is a wild-type or optimized tag protein.

In another preferred embodiment, the tag protein is selected from the group consisting of: streptavidin, MBP, GST, Protein, CBP, or a combination thereof.

In another preferred embodiment, the tag protein is optimized streptavidin.

In another preferred embodiment, the tag protein carries a Biotin label.

In another preferred embodiment, the tag protein has (Kd) of not more than 10^-6mol/L，(Kd<＝10^-6mol/L); or at 10^-6mol/L to 10^-17Binding constants of encoded proteins between mol/L to Biotin.

In another preferred embodiment, the coding sequence of the tag protein is selected from the group consisting of;

(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 2 (amino acid sequence of streptavidin: EAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNA HSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS);

(b) a polynucleotide having a sequence as shown in SEQ ID No. 1 or 4;

(c) polynucleotides having a nucleotide sequence homology of 30% or more, preferably 75% (preferably 85% or more, more preferably 90% or more or 95% or more or 98% or more or 99%) to the sequence shown in SEQ ID No. 1 or 4;

(d) a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID NO. 1 or 4;

(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).

In another preferred example, the tag 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 30% homology (preferably, more than or equal to 85% homology, more preferably, more than or equal to 90% homology, more preferably, more than or equal to 95% homology, and most preferably, more than or equal to 97% homology, such as more than 98% or more than 99%) with the amino acid sequence shown in SEQ ID NO. 2 and has the same activity as the sequence shown in SEQ ID NO. 2.

In another preferred embodiment, the linker sequence is a codon optimized linker sequence.

In another preferred embodiment, the linker sequence has a sequence that is not prone to secondary structure formation (e.g., AT-rich sequence, hairpin-free sequence, G-quadruplex-free sequence, etc.), and is not rich in rare codons.

In another preferred embodiment, the linker sequence may have an enzyme cleavage site.

In another preferred embodiment, the linker sequence is selected from the group consisting of;

(i) a polynucleotide having a sequence as set forth in SEQ ID No. 3 or 5;

(ii) polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95% or more or 98% or more or 99%) with the sequence shown in SEQ ID No. 3 or 5;

(iii) a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO. 3 or 5;

(iv) (iv) a polynucleotide complementary to any one of the polynucleotides of (i) to (iii).

In another preferred embodiment, the linker sequence is as shown in SEQ ID No. 3 or 5.

In another preferred embodiment, the coding sequence of the foreign protein is from a prokaryote or a eukaryote.

In another preferred embodiment, the coding sequence of the foreign protein is from an animal, plant, pathogen.

In another preferred embodiment, the coding sequence for the foreign protein is from a mammal, preferably a primate, a rodent, including a human, a mouse, a rat.

In another preferred embodiment, the coding sequence of the foreign protein is selected from the group consisting of: exogenous DNA encoding a luciferin protein, or a luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyltrna synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, a variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.

In another preferred embodiment, the foreign protein is selected from the group consisting of: luciferin protein, or luciferase (e.g., firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of an antibody, luciferase mutation, alpha-amylase, enterobactin 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 embodiment, the nucleic acid construct further comprises a promoter upstream of the 5' end.

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

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

In another preferred embodiment, the enhancer element comprises an IRES element, an RBS element, a non-coding sequence, or a combination thereof.

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

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

In another preferred embodiment, the IRES element comprises an endogenous IRES element and an exogenous IRES element.

In another preferred embodiment, the IRES element is derived from one or more cells selected from the group consisting of: human (human), Chinese hamster ovary Cells (CHO), insect cells (instect), Wheat germ (Wheat cells), Rabbit reticulocyte (Rabbit reticulocyte).

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

In a second aspect, the invention provides a vector or combination of vectors comprising a nucleic acid construct according to the first aspect of the invention.

In a third aspect, the invention provides a genetically engineered cell having a construct according to the first aspect integrated at one or more sites in its genome or comprising a vector or combination of vectors according to the first aspect of the invention.

In another preferred embodiment, the genetically engineered cell comprises a prokaryotic cell and a eukaryotic cell.

In another preferred embodiment, the genetically engineered cell is selected from the group consisting of: human cells (e.g., Hela cells), chinese hamster ovary cells, insect cells, wheat germ cells, rabbit reticulocyte, yeast cells, or combinations thereof.

In another preferred embodiment, the genetically engineered cell is a yeast cell.

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 marxianus, Kluyveromyces polybracteus, or a combination thereof.

In a fourth aspect, the present invention provides a kit comprising reagents selected from one or more of the following groups:

(a) the construct of the first aspect of the invention;

(b) a vector or combination of vectors according to the second aspect of the invention; and

(c) the genetically engineered cell according to the third aspect of the invention.

In another preferred embodiment, the kit further comprises (d) a eukaryotic in vitro biosynthesis system (e.g., a eukaryotic in vitro protein synthesis system).

In another preferred embodiment, the eukaryotic in vitro biosynthetic system is selected from the group consisting of: a yeast in vitro biosynthesis system, a chinese hamster ovary cell in vitro biosynthesis system, an insect cell in vitro biosynthesis system, a Hela cell in vitro biosynthesis system, or a combination thereof.

In another preferred embodiment, the eukaryotic in vitro biosynthetic system comprises a eukaryotic in vitro protein synthetic system.

In another preferred embodiment, the eukaryotic in vitro protein synthesis system is selected from the group consisting of: a yeast in vitro protein synthesis system, a chinese hamster ovary cell in vitro protein synthesis system, an insect cell in vitro protein synthesis system, a Hela cell in vitro protein synthesis system, or a combination thereof.

In another preferred embodiment, the kit further comprises (e) a yeast in vitro biosynthesis system (e.g., a yeast in vitro protein synthesis system).

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

In a fifth aspect, the invention provides a construct according to the first aspect, a vector or a combination of vectors according to the second aspect, a genetically engineered cell according to the third aspect, or a kit according to the fourth aspect, for use in performing high-throughput in vitro protein synthesis.

The sixth aspect of the present invention provides an in vitro high-throughput method for synthesizing a foreign protein, comprising the steps of:

(i) providing a nucleic acid construct according to the first aspect of the invention in the presence of a eukaryotic in vitro biosynthesis system;

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

In another preferred example, the method further comprises: (iii) optionally isolating or detecting said foreign protein from said eukaryotic in vitro biosynthetic system.

In another preferred embodiment, the eukaryotic in vitro biosynthesis system is a yeast in vitro biosynthesis system (e.g., a yeast in vitro protein synthesis system).

In another preferred embodiment, the coding sequence of the foreign protein encodes a foreign protein selected from the group consisting of: luciferin protein, or luciferase (e.g., firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of an antibody, luciferase mutant, alpha-amylase, enteromycin 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 embodiment, in the step (ii), the reaction temperature is 20 to 37 ℃, preferably 22 to 35 ℃.

In another preferred embodiment, in the step (ii), the reaction time is 1 to 10 hours, preferably 2 to 8 hours.

In a seventh aspect, the invention provides a linker sequence selected from the group consisting of:

(i) a polynucleotide having a sequence as set forth in SEQ ID No. 3 or 5;

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 the basic biological process from DNA to protein.

FIG. 2 shows Relative Fluorescence unit values (RFUs) of Enhanced green Fluorescence protein (eGFP) synthesized by 2 eukaryotic Streptavidin sequences initiated in vitro protein synthesis system; STN1 (natural Streptavidin gene sequence is directly retrieved from a gene bank) and STN2 (partial bases are modified by adopting a synonymous codon replacing mode according to the characteristics of the whole sequence under the premise of not changing the amino acid sequence) are connected with the enhanced green fluorescent protein through Linker2 to form STN-Linker (named SAN); the relative fluorescence unit values of the two reach more than 400, wherein, the relative fluorescence unit value of reaction for 20 hours reaches 570 and 620 respectively, NC (negative control) is the reading value of an experimental group without adding a nucleic acid construct, and the protein expression quantity can be purified.

FIG. 3 shows Relative Fluorescence unit values (RFUs) of Enhanced green fluorescent protein (eGFP) initially synthesized in the yeast in vitro protein synthesis system using different Linker sequences for STN1 and the target protein (eGFP), NC being the reading of the experimental group without the addition of the nucleic acid construct, showing that the Relative Fluorescence unit values were significantly improved using modified linkers (Linker2 and Linker 3).

FIG. 4 shows that biotin beads were added to the reaction solution to bind the target foreign protein, and SDS-PAGE protein gel detection was performed, which indicated that the band size was correct and clearly visible. STN1 and STN2 refer to experimental groups in which the target protein is not linked after STN (STN1 and STN2), and SAN1-eGFP and SAN2-eGFP correspond to experimental groups in which the target protein (eGFP) is linked after STN (STN1 and STN2), respectively.

FIG. 5 shows the basic principle of SAN applied to protein expression and purification, after SAN and target protein coding sequence are transcribed and translated, the target protein is grabbed in the reaction solution by specific binding with biotin beads. To separate the target protein and SAN fragment, an enzyme cleavage reaction can be performed by inserting an enzyme cleavage site (e.g., TEV cleavage site) between the two fragments.

Detailed Description

As a result of extensive and intensive studies, it has been unexpectedly found for the first time, through extensive screening and search, that a nucleic acid construct containing a Streptavidin-Linker sequence useful for the expression and purification of a target protein, which is composed of an optional 5' UTR (e.g., promoter, translation initiation site, yeast-derived IRES enhancer, ScGPR1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1, etc.), a Streptavidin sequence useful for the expression and purification of a target protein, a Linker (SAN) (preferably a codon-optimized Linker or an optimized Linker containing an enzyme cleavage site) linking the Streptavidin sequence and a coding sequence of a foreign protein, and a coding sequence and 3 UTR of a foreign protein, can be accomplished by applying the nucleic acid construct of the present invention to a protein synthesis system (particularly an in vitro protein synthesis system) of the present invention

Protein synthesis system

Protein synthesis refers to the process by which an organism synthesizes a protein according to genetic information on messenger ribonucleic acid (mRNA) transcribed from deoxyribonucleic acid (DNA). Protein biosynthesis is also known as Translation (Translation), the process by which the sequence of bases in an mRNA molecule is converted into the sequence of amino acids in a protein or polypeptide chain. This is the second step in gene expression, the final stage in the production of the gene product protein. Different tissue cells have different physiological functions because they express different genes to produce proteins with specific functions, and more than 200 components involved in protein biosynthesis are mainly composed of mRNA, tRNA, ribosomes, and related enzymes and protein factors.

The in vitro protein synthesis system is generally characterized in that mRNA or DNA template, RNA polymerase, amino acid, ATP and other components are added into a lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of exogenous proteins. Currently, commercial in vitro protein expression systems that are frequently tested include the e.coli system (ECE), Rabbit Reticulocyte Lysate (RRL), Wheat Germ (WGE), Insect Cell Extract (ICE) and human-derived systems. Compared with the traditional in vivo recombinant expression system, the in vitro cell-free synthesis system has multiple advantages, such as the capability of expressing special proteins with toxic action on cells or containing unnatural amino acids (such as D-amino acids), capability of directly taking PCR products as templates to simultaneously synthesize multiple proteins in parallel and development of high-throughput drug screening and proteomics research.

Among them, yeast (yeast) has 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 yeasts. 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, a preferred protein synthesis system is an in vitro protein synthesis system, and in the present invention, the in vitro protein synthesis system is not particularly limited, and a preferred in vitro protein synthesis system is a Kluyveromyces expression system (more preferably, a Kluyveromyces lactis expression system).

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

(a) a yeast cell extract;

(b) polyethylene glycol;

(c) optionally exogenous sucrose; and

(d) 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 mixtures, phosphocreatine, Dithiothreitol (DTT), phosphocreatine kinase, rnase inhibitors, fluorescein, 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 cell extract in the in vitro protein synthesis system is not particularly limited, and usually the 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 cell extract does not contain intact cells, and typical cell extracts include ribosomes for protein translation, transfer RNAs, aminoacyl tRNA synthetases, initiation and elongation factors required for protein synthesis, and stop release factors. In addition, the cell extract also contains some other proteins, especially soluble proteins, which originate from the cytoplasm of the cell.

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

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

(i) providing a cell;

(ii) washing the cells to obtain washed cells;

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

(iv) and carrying out solid-liquid separation on the cell crude extract to obtain a liquid part, namely the cell 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 cell disruption treatment is not particularly limited, and a preferred cell disruption treatment includes high pressure disruption, freeze-thaw (e.g., liquid nitrogen low temperature) disruption.

The nucleoside triphosphate mixture in the in vitro protein synthesis system is adenosine triphosphate, guanine nucleoside 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 0.02-0.2 mM, 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 generally, 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 a cell 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 creatine phosphate, 1.7mM of dithiothreitol, 0.27mg/mL of phosphocreatine kinase, 1% -4% of polyethylene glycol, 0.5% -2% of sucrose, 8-20 ng/microliter of DNA of firefly luciferase and 0.027-0.054mg/mL of T7RNA polymerase.

Coding sequence of foreign protein (foreign DNA)

As used herein, the term "coding sequence for a foreign protein" is used interchangeably with "foreign DNA" and refers to a foreign DNA molecule used to direct protein synthesis. Typically, the DNA molecule is linear or circular. The DNA molecule contains a sequence encoding a foreign protein.

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

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

The foreign DNA may also be selected from the group consisting of: exogenous DNA encoding alpha-amylase, enteromycin 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 (eGFP), Yellow Fluorescent Protein (YFP), escherichia coli β -galactosidase (lactasise, LacZ), human Lysine-tRNA synthetase (Lysine-tRNA synthetase), human Leucine-tRNA synthetase (Leucine-tRNA synthetase), arabidopsis thaliana Glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase), murine Catalase (Catalase), or combinations thereof.

Nucleic acid constructs

Z1-Z2-Z3 (I)

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

z1, Z2, Z3 are the elements used to construct the construct, respectively;

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

z1 is the coding sequence of the tag protein;

z2 is a linker sequence;

z3 is the coding sequence of no or foreign protein.

As a result of extensive and intensive studies, it has been unexpectedly found for the first time, through extensive screening and search, that a nucleic acid construct containing a Streptavidin-Linker sequence useful for the expression and purification of a target protein, which is composed of an optional 5' UTR (e.g., promoter, translation initiation site, yeast-derived IRES enhancer, ScGPR1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1, etc.), a Streptavidin sequence useful for the expression and purification of a target protein, a Linker (SAN) (preferably a codon-optimized Linker or an optimized Linker containing an enzyme cleavage site) linking the Streptavidin sequence and a coding sequence of a foreign protein, and a promoter and a 3 UTR of a foreign protein, is used in a protein synthesis system (particularly an in vitro protein synthesis system) of the present invention, and the expression and purification of a foreign protein can be accomplished, and the RFU value of the synthesized foreign protein can be as high as 600 or more, compared with a nucleic acid construct without SAN sequence, the improvement is 2 times. On this basis, the present inventors have completed the present invention.

In the present invention, the selection of the coding sequence of the foreign protein is not particularly limited, and generally, the coding sequence of the foreign protein is selected from the group consisting of: exogenous DNA encoding a luciferin protein, or luciferase (e.g., firefly luciferase), a green fluorescent protein, a yellow fluorescent protein, an aminoacyltrna synthetase, a glyceraldehyde-3-phosphate dehydrogenase, a catalase, a myokinetin, a variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.

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

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

In a preferred embodiment, the nucleic acid construct of the present invention is represented in sequence listing 1.

In another preferred embodiment, said construct further comprises an element or a combination thereof selected from the group consisting of: promoters, terminators, poly (A) elements, transport elements, gene targeting elements, selectable marker genes, enhancers, resistance genes, transposase encoding genes.

A variety of selectable marker genes are applicable to the present invention, including but not limited to: auxotrophic markers, resistance markers, reporter gene markers. The use of a selectable marker plays a role in the screening of recombinant cells (recombinants) so that recipient cells can be significantly distinguished from non-transformed cells. The auxotrophic marker is a marker gene that is transformed so as to complement a mutant gene of a recipient cell, thereby allowing the recipient cell to exhibit wild-type growth. The resistance marker refers to transferring resistance genes into receptor cells, and the transferred genes enable the receptor cells to show drug resistance at a certain drug concentration. As a preferred mode of the invention, resistance markers are used to facilitate the selection of recombinant cells.

In the invention, the nucleic acid construction is applied in the in-vitro protein synthesis system, and after the reaction is finished, the biotin-binding technology is used, so that the expression and purification of foreign protein can be finished, and particularly, the relative light unit value of the quantity of the enhanced green fluorescent protein synthesized by the nucleic acid construction is relatively high.

Vector, genetically engineered cell

The invention also provides a vector or combination of vectors comprising the nucleic acid construct of the invention. Preferably, the vector is selected from: bacterial plasmids, bacteriophages, yeast plasmids, or animal cell vectors, shuttle vectors; the vector is a transposon vector. Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. Any plasmid and vector may be used as long as it can replicate and is stable in the host.

One of ordinary skill in the art can use well-known methods to construct expression vectors containing the promoter and/or gene sequences of interest described herein. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

The invention also provides a genetic engineering cell, wherein the genetic engineering cell contains the construct or the vector combination, or the chromosome of the genetic engineering cell is integrated with the construct or the vector. In another preferred embodiment, the genetically engineered cell further comprises a vector comprising a gene encoding a transposase or having a transposase gene integrated into its chromosome.

Preferably, the genetically engineered cell is a eukaryotic cell.

In another preferred embodiment, the eukaryotic cell includes (but is not limited to): a yeast cell (preferably, a kluyveromyces cell, more preferably a kluyveromyces lactis cell).

The constructs or vectors of the invention may be used to transform appropriate genetically engineered cells. The genetically engineered cells may be prokaryotic cells, such as E.coli, Streptomyces, Agrobacterium: or lower eukaryotic cells, such as plant cells, yeast cells; or higher eukaryotic cells such as insect cells, animal cells, etc. It is clear to one of ordinary skill in the art how to select appropriate vectors and genetically engineered cells. Transformation of genetically engineered cells with recombinant DNABy conventional techniques well known to those skilled in the art. When the host is prokaryote (e.g., Escherichia coli), CaCl may be used₂The treatment can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). The transformed plant may be transformed by methods such as Agrobacterium transformation or biolistic transformation, for example, leaf disc method, immature embryo transformation, flower bud soaking method, etc.

In vitro high-flux protein synthesis method

The invention provides an in vitro high-flux protein synthesis method, which comprises the following steps:

(i) providing a nucleic acid construct according to the first aspect of the invention in the presence of an in vitro protein synthesis system;

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

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

The main advantages of the invention include:

(1) the invention discovers for the first time that optional promoter, ribosome binding site, IRES, STN (STN1, STN2) sequence for expression and purification of target protein and coding sequence of foreign protein are used as nucleic acid construction, and the nucleic acid construction can be applied to the in vitro protein synthesis system of the invention and can be used for expression and purification of foreign protein.

(2) The Streptavidin-linker (SAN) amino acid sequence disclosed by the invention can be specifically identified and efficiently combined with beads. The recognition and combination efficiency of the amino acid sequences not only exceeds that of the traditional specific amino acid sequences, but also can be applied to an in vitro protein synthesis system of a yeast source (especially a Kluyveromyces lactis source).

(3) Compared with other cells, the kluyveromyces lactis can be applied to the production of proteins in the fields of food and medicines due to the safety and high efficiency of the kluyveromyces lactis, and the in vitro protein synthesis system has the advantages of suitability for high-throughput protein synthesis screening, toxic protein synthesis, short time, low cost and the like, so the in vitro protein synthesis system derived from the kluyveromyces lactis cells can be widely applied to related fields.

(4) The SAN amino acid sequence provided by the invention not only can improve the expression and purification effects of target foreign proteins, but also can increase the possibility of synthesizing different proteins in a Kluyveromyces lactis in-vitro protein synthesis system.

(5) The invention discloses a nucleic acid construct consisting of Streptavidin-Linker sequences which can be used for expression and purification of target proteins for the first time, the expression and purification of foreign proteins can be completed by applying the nucleic acid construct in a protein synthesis system (especially an in-vitro protein synthesis system) of the invention, and the RFU value of the synthesized foreign proteins can be up to more than 600, and is improved by 2 times compared with the nucleic acid construct without SAN sequences and is improved by 3 times compared with the nucleic acid construct containing non-optimized linkers.

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.

The foreign protein is exemplified by eGFP.

EXAMPLE 1 eukaryotic Streptavidin amino acid sequence determination

1.1 determination of Streptavidin amino acid sequence: related nucleic acid sequences and corresponding amino acid sequences are found from a gene bank and used as corresponding biotin sequences, the WtStreptavidin sequence (STN1) is a natural streptavidin sequence directly called from the gene bank, and the OurStreptavidin sequence (STN2) modifies partial bases by adopting a mode of synonymous codon substitution according to the characteristics of the whole sequence on the premise of not changing the amino acid sequences. OurStreptavidin and WildtypeStreptavidin, corresponding to the same amino acid sequence. The sequence determination of Linker depends on that the amino acid sequence is similar to natural protein as much as possible, the homology of the corresponding amino acid sequence is not less than 30%, and the nucleic acid sequence does not contain strong secondary structure sequence, hairpin structure, G-quadruplex and the like (the GC content is not fixed). The Linker3 contains a TEV enzyme cutting site sequence, and can be used for obtaining pure target protein after enzyme cutting.

TABLE 1 nucleic acid sequences related to Kluyveromyces lactis

EXAMPLE 2 construction of in vitro protein Synthesis System plasmids containing eukaryotic Streptavidin nucleic acid sequences

2.1 Total Gene Synthesis: the artificially designed 2 STN nucleic acid sequences (STN1, STN2) were ligated in tandem and synthesized by whole-gene synthesis.

2.2 construction of plasmid: two primer pairs, with the suffix 1f and 1b, were used for the artificially designed 2 STN nucleic acid sequences (STN1, STN2) to amplify the corresponding STN fragments from the synthesized DNA fragments, respectively, and the primers with the suffix 2f and 2b were used for the PCR amplification of the pD2P-eGFP plasmid. In the final constructed plasmid, 2 STN nucleic acid sequences (STN1, STN2) were inserted between the AUG start codon and eGFP of the pD2P-eGFP plasmid. The names of the plasmids are: pD2P-STN2_ eGFP and pD2P-STN1_ eGFP.

The Linker2 sequence was inserted into the existing plasmid pD2P-STN _ eGFP by PCR method, inserted between the STN (STN1, STN2) nucleic acid sequence and the foreign protein coding sequence (eGFP), to construct plasmids pD2P-SAN1_ eGFP and pD2P-SAN2_ eGFP containing STN-Linker (SAN), respectively, the name and sequence number of which are listed in Table 2.

The specific construction process is as follows:

for plasmid construction of pD2P-STN _ eGFP, PCR amplification was performed using two pairs of primers, respectively, and 10. mu.L each of the amplification products was mixed; adding 1 mu L of Dpn I into 20 mu L of the amplification product, and incubating for 6h at 37 ℃; adding 4 mu L of the product treated by the DpnI into 50 mu L of DH5 alpha competent cells, placing on ice for 30min, thermally shocking at 42 ℃ for 45s, placing on ice for 3min, adding 200 mu L of LB liquid culture medium, performing shake culture at 37 ℃ for 4h, and coating on LB solid culture medium containing Amp antibiotics for overnight culture; 6 monoclonals are picked for amplification culture, sequencing is carried out to confirm correctness, and plasmids are extracted for storage.

TABLE 2 primer sequences

Example 3 use of STN nucleic acid sequences in vitro protein Synthesis systems

3.1 amplification of a fragment comprising the SAN sequence between the transcription start and stop sequences of T7 and a fragment of pD2P-eGFP in all plasmids by means of the PCR method using the primers pD2P _ F: CGCGAAATTAATACGACTCACTATAGG (SEQ ID NO.:16 and pD2P _ R: TCCGGATATAGTTCCTCCTTTCAG (SEQ ID NO.: 17).

And purifying and enriching the amplified DNA fragment by using an ethanol precipitation method: adding 1/10 volume of 3M sodium acetate (pH5.2) into the PCR product, adding 2.5-3 times volume of 95% ethanol, and incubating on ice for 15 min; centrifuging at room temperature at a speed higher than 14000g for 30min, and discarding the supernatant; washing with 70% ethanol, centrifuging for 15min, discarding the supernatant, dissolving the precipitate with ultrapure water, and determining the DNA concentration.

3.2 the purified DNA fragments were added to the home-made in vitro protein synthesis system according to the instructions. And placing the reaction system in an environment with the temperature of 22-30 ℃, 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 (Perkin Elmer), and read to detect the intensity of eGFP signal and Relative light Unit value (RFU) as an activity Unit, as shown in fig. 1.

3.3 adding biotin-beads to the reaction solution to bind the target foreign protein, and performing SDS-PAGE protein gel detection, as shown in FIG. 2.

Results of the experiment

1. Construction of plasmid for in vitro protein Synthesis System

Through multiple attempts, successfully constructed 4 extrasomatic protein synthesis system plasmids including all 2 plasmids containing eGFP detection protein and 2 plasmids without eGFP detection protein (plasmid names pD2P-SAN1_ eGFP, pD2P-SAN2_ eGFP, pD2P-STN1 and pD2P-STN2) were finally constructed.

Use of SAN sequences in vitro protein Synthesis systems

As shown in FIG. 2, the Relative Fluorescence Units (RFU) emitted by the enhanced green fluorescent protein in the in vitro protein synthesis system due to the 2 SAN sequences screened all reached higher values (RFU values reached above 400 after 3 hours of reaction), which were comparable to the RFU value of the nucleic acid construct containing linker3, and increased by 3-fold over the RFU value of the nucleic acid construct containing linker1.5 (RFU of the nucleic acid construct containing linker1.5 was only 60% of the RFU of the foreign protein of the nucleic acid construct containing linker 2), and protein purification experiments were possible.

The results of the invention show that: the SAN sequence can be used for detecting the protein expression and purification effect, and can be applied to an in vitro protein synthesis system, and the efficiency of initial protein synthesis can reach or exceed that of a commonly used tag sequence. The selectivity of the protein expression and purification mode of the in vitro synthesis system is increased, and the availability of the in vitro protein synthesis system is greatly enhanced.

In addition, the research of the invention also finds that the combination of 5 '-UTR with simple structure and high efficiency, strong promoters (such as T7 promoter, T3 promoter and SP6 promoter), different IRES elements (such as KLNCE102) and different STN sequences, 3' -UTR and the like can also obtain very good protein expression and purification effects, wherein the RFU value of the target protein (eGFP) is as high as over 600, and the relative fluorescence unit value (RFU) is improved by 2 times compared with that without SAN sequence.

Comparative example

In the nucleic acid construct of the present invention, the RFU of the foreign protein is 85 without a linker.

All documents referred to herein are incorporated by reference into this application as if each had been 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.Garcia RA,Riley MR.Applied biochemistry and biotechnology.Humana Press, 1981.263-264.

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

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

4.

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

5.

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

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

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

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

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

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

Sequence listing

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Claims

1. A nucleic acid construct having from 5 'to 3' a structure of formula I:

Z1-Z2-Z3 (I)

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

z1, Z2, Z3 are the elements used to construct the construct, respectively;

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

z1 is a coding sequence of a tag protein, the tag protein is streptavidin, and the sequence of the streptavidin is shown in SEQ ID NO. 1 or SEQ ID NO. 4;

z2 is a connecting sequence, and Z2 is selected from the following group:

(i) the polynucleotide with the sequence as shown in SEQ ID No. 3 or 5; and

(ii) (ii) a polynucleotide complementary to the polynucleotide of (i);

z3 is the coding sequence of no or foreign protein.

2. The nucleic acid construct of claim 1, wherein Z3 is a coding sequence for enhanced green fluorescent protein.

3. A vector or vector combination comprising the nucleic acid construct of claim 1.

4. A genetically engineered cell having the nucleic acid construct of claim 1 integrated at one or more sites in the genome of the genetically engineered cell, or having the vector or combination of vectors of claim 3 incorporated therein.

5. A kit comprising reagents selected from one or more of the group consisting of:

(a) the nucleic acid construct of claim 1;

(b) the vector or combination of vectors of claim 3; and

(c) the genetically engineered cell of claim 4.

6. Use of the nucleic acid construct of claim 1, the vector or combination of vectors of claim 3, the genetically engineered cell of claim 4, or the kit of claim 5 for high-throughput in vitro protein synthesis.

7. An in vitro high-flux synthesis method of exogenous protein is characterized by comprising the following steps:

(i) providing the nucleic acid construct of claim 1 in the presence of a eukaryotic in vitro biosynthesis system;

8. The method of claim 7, wherein the method further comprises: (iii) isolating or detecting the foreign protein from the eukaryotic in vitro biosynthetic system.

9. A linker sequence selected from the group consisting of seq id nos:

(i) the polynucleotide with the sequence as shown in SEQ ID No. 3 or 5; and

(ii) (ii) a polynucleotide complementary to the polynucleotide of (i).