DNA element for enhancing cell-free expression protein and application of DNA element in D2P

Abstract

The invention provides a DNA element for enhancing cell-free expression protein and application of the DNA element in D2P, and particularly, by optimizing a 3-UTR sequence, enhancing the yield of the cell-free expression protein and saving time cost and capital cost, commercial application of the protein in the fields of scientific research, medicine, massive health, beauty, food and the like is greatly promoted.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a DNA element for enhancing cell-free protein expression and an application thereof and an application thereof in D2P. Background Art

[0002] Protein is the material basis of all life and an important component of body cells. Protein has important applications and great value in the fields of biology and medicine. For example, molecular enzymes (Taq enzymes, Pfu enzymes), antibodies, hormones, hemoglobin, collagen, sweet protein, etc. are all proteins. These proteins have unlimited commercial value in scientific research, medicine, health, beauty, food and other fields. Protein has such a huge application and prospect. How to prepare these proteins in large quantities at a lower cost is an imminent technical problem. Traditional methods generally express and purify through Escherichia coli, yeast cells, insect cells, and mammalian cells. In the fields of applied medicine and food, Escherichia coli faces the problem of removing endotoxins. However, the production cycle of yeast cells, insect cells, and mammalian cells is relatively long and the cost is high.

[0003] In addition to the above understanding of protein synthesis in cells, protein synthesis can also be carried out outside cells. The in vitro protein synthesis system generally refers to the addition of mRNA or DNA templates, RNA polymerase, amino acids, ATP and other components to the lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of exogenous proteins.

[0004] In the process of in vitro protein synthesis, the structure of mRNA has a particularly great influence on the protein expression, for example, the 3-UTR sequence structure. However, many studies on these sequence structures are limited to the field of cell-synthesized proteins, and there are huge differences between cell-synthesis and cell-free synthesis. This results in that sequences that are effective in cell synthesis are not necessarily effective for cell-free system synthesis. Therefore, there is an urgent need in the art to develop a genetically modified mRNA that can enhance protein expression, thereby further reducing the cost of protein synthesis. Summary of the invention

[0005] The purpose of the present invention is to improve the protein expression level of a cell-free protein expression (DNA to Protein, D2P) system through genetic modification.

[0006] The purpose of the present invention is to provide a nucleic acid construct that can be used for protein expression and purification in eukaryotic cells. The application of the nucleic acid construct in an in vitro protein expression system can improve the expression efficiency of foreign proteins and reduce costs.

[0007] The first aspect of the present invention provides a nucleic acid construct, wherein the nucleic acid construct has a structure of formula I from 5' to 3':

[0008] Z1-Z2(I)

[0009] In the formula,

[0010] Z1 and Z2 are respectively elements used to constitute the structure;

[0011] Each "-" is independently a bond or a nucleotide linking sequence;

[0012] Z1 is the coding sequence of no or foreign protein;

[0013] Z2 is a nucleic acid sequence without or without a 3' untranslated region (3-UTR),

[0014] The Z2 and Z1 are modified sequences of the wild-type sequence;

[0015] The nucleic acid construct is capable of enhancing cell-free expression of a protein.

[0016] The term “enhancement” refers to increasing the efficiency and / or yield of cell-free protein expression by at least 5%, preferably by more than 10%, more preferably by more than 30%, and most preferably by more than 100%.

[0017] Furthermore, the connection sequence indicated by "-" is a codon-optimized connection sequence.

[0018] In another preferred embodiment, the linker sequence has a sequence that is not easy to form a secondary structure (such as an AT-rich sequence, no hairpin structure, no G-quadruplex, etc.), and is not rich in rare codons.

[0019] In another preferred embodiment, the linker sequence may have a restriction enzyme cleavage site.

[0020] Furthermore, the coding sequence of the exogenous protein represented by Z1 comes from prokaryotes or eukaryotes.

[0021] In another preferred embodiment, the coding sequence of the exogenous protein comes from animals, plants, or pathogens.

[0022] In another preferred embodiment, the coding sequence of the exogenous protein is from mammals, preferably primates, rodents, including humans, mice, and rats.

[0023] In another preferred embodiment, the exogenous protein is selected from the group consisting of a soluble protein, a functional protein, and a natural protein or a mutant protein or an artificially designed protein of an active protein.

[0024] In another preferred embodiment, the coding sequence of the exogenous protein is selected from the following group: exogenous DNA encoding luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody variable region, luciferase mutant DNA or a combination thereof.

[0025] In another preferred embodiment, the exogenous protein is selected from the following group: luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.

[0026] In another preferred embodiment, the coding sequence of the 3' untranslated region (3-UTR) represented by Z2 is selected from one of the following groups;

[0027] (a) a polynucleotide as shown in any one of SEQ ID NO.145 to SEQ ID NO.171;

[0028] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.145 to SEQ ID NO.171;

[0029] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 145 to SEQ ID NO. 171;

[0030] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0031] In a preferred example, the nucleic acid construct has a structure of Formula I from 5' to 3':

[0032] Z3-Z1-Z2(I)

[0033] In the formula,

[0034] Z3, Z1, and Z2 are respectively elements used to constitute the structure;

[0035] Each "-" is independently a bond or a nucleotide linking sequence;

[0036] Z3 is the coding sequence of the signal peptide;

[0037] Z1 is the coding sequence of no or foreign protein;

[0038] Z2 is the nucleic acid sequence of the 3' untranslated region (3-UTR),

[0039] The Z3 and Z2 are modified sequences of the wild-type sequence;

[0040] The nucleic acid construct is capable of enhancing cell-free expression of a protein.

[0041] The Z3 is a signal peptide coding sequence source selected from the group consisting of one or more cells selected from the group consisting of prokaryotic cells and eukaryotic cells. In another preferred embodiment, the signal peptide source is selected from the group consisting of one or more cells selected from the group consisting of human, Chinese hamster ovary cells (CHO), insect cells, wheat germ cells, rabbit reticulocytes, and yeast cells.

[0042] The nucleic acid construct further comprises a coding sequence of a signal peptide, wherein the amino acid sequence of the signal peptide is selected from one of the following groups:

[0043] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.72;

[0044] In another preferred embodiment, the coding sequence of the signal peptide is selected from one of the following groups;

[0045] (a) a polynucleotide represented by any one of SEQ ID NO.73 to SEQ ID NO.144;

[0046] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.73 to SEQ ID NO.144;

[0047] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 73 to SEQ ID NO. 144;

[0048] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0049] Furthermore, the nucleic acid construct further comprises an enhancer element, a Kozak sequence, an Ω sequence, or a combination thereof.

[0050] Furthermore, the IRES element is derived from one or more cells selected from the following groups: human, Chinese hamster ovary cell (CHO), insect cell, wheat germ cell, rabbit reticulocyte.

[0051] In another preferred embodiment, the enhancer element is an IRES element. Further preferably, the IRES element is selected from one of the following groups:

[0052] (a) a polynucleotide as shown in any one of SEQ ID NO.172 to SEQ ID NO.248;

[0053] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.172 to SEQ ID NO.248;

[0054] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 172 to SEQ ID NO. 248;

[0055] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0056] In another preferred embodiment, the kozak sequence is selected from one of the following groups:

[0057] (a) a polynucleotide as shown in any one of SEQ ID NO.249 to SEQ ID NO.254;

[0058] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to the sequence shown in any one of SEQ ID NO.249 to SEQ ID NO.254;

[0059] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 249 to SEQ ID NO. 254;

[0060] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0061] In another preferred embodiment, the Ω sequence is selected from one of the following groups:

[0062] (a) a polynucleotide as shown in any one of SEQ ID NO.255 ​​to SEQ ID NO.269;

[0063] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.255 ​​to SEQ ID NO.269;

[0064] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 255 to SEQ ID NO. 269;

[0065] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0066] In another preferred embodiment, the IRES element, the kozak sequence and the Ω sequence are combined to form a 5' untranslated region (5-UTR).

[0067] In another preferred embodiment, the 5' untranslated region (5-UTR) is selected from one of the following groups:

[0068] (a) a polynucleotide as shown in any one of SEQ ID NO.270 to SEQ ID NO.298;

[0069] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.270 to SEQ ID NO.298;

[0070] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 270 to SEQ ID NO. 298;

[0071] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0072] In another preferred embodiment, the nucleic acid construct has a structure of Formula II from 5' to 3':

[0073] Y1-Y2-Y3-Z3-Z1-Z2(I)

[0074] In the formula,

[0075] Y1, Z3, Z1, and Z2 are respectively elements used to constitute the construct;

[0076] Each "-" is independently a bond or a nucleotide linking sequence;

[0077] Z3 is the coding sequence of the signal peptide;

[0078] Z1 is the coding sequence of no or foreign protein;

[0079] Z2 is the nucleic acid sequence of the 3' untranslated region (3-UTR);

[0080] Y1 is absent or is an enhancer element, preferably an IRES element;

[0081] Y2 is none or a Kozak sequence;

[0082] Y3 is None or an Ω sequence.

[0083] Furthermore, the 5' end upstream of the nucleic acid construct also includes a promoter.

[0084] In another preferred embodiment, the promoter is selected from one of the following groups: T7 promoter, T3 promoter, SP6 promoter, or a combination thereof. More preferably, the promoter is T7 promoter.

[0085] Furthermore, the 3' end downstream of the nucleic acid construct also includes a terminator.

[0086] In another preferred embodiment, the terminator is selected from one of the following groups: T7 terminator, T3 terminator, SP6 terminator, or a combination thereof.

[0087] In another preferred embodiment, the terminator sequence is selected from one of the following groups:

[0088] (a) encoding a polynucleotide as shown in any one of SEQ ID NO.300 to SEQ ID NO.304;

[0089] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.300 to SEQ ID NO.304;

[0090] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 300 to SEQ ID NO. 304;

[0091] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0092] In another preferred embodiment, the nucleic acid construct has a structure of Formula II from 5' to 3':

[0093] X1-Y1-Y2-Y3-Z3-Z1-Z2-X2(I)

[0094] In the formula,

[0095] Y1, Z3, Z1, and Z2 are respectively elements used to constitute the construct;

[0096] Each "-" is independently a bond or a nucleotide linking sequence;

[0097] Z3 is the coding sequence of the signal peptide;

[0098] Z1 is the coding sequence of no or foreign protein;

[0099] Z2 is the nucleic acid sequence of the structure-optimized 3' untranslated region (3-UTR);

[0100] Y1 is absent or is an enhancer element, preferably an IRES element;

[0101] Y2 is none or a Kozak sequence;

[0102] Y3 is none or an Ω sequence;

[0103] X1 is the none or promoter series;

[0104] X2 is none or the terminator series.

[0105] In another preferred embodiment, the construct further comprises an element or a combination thereof selected from the following groups: a poly(A) element, a transport element, a gene targeting element, a selection marker gene, an enhancer, a resistance gene, a transposase encoding gene, etc.

[0106] The second aspect of the present invention provides a vector or a vector combination, wherein the vector or the vector combination contains the nucleic acid construct described in the first aspect of the present invention.

[0107] The third aspect of the present invention provides a genetically engineered cell, wherein one or more sites of the genome of the genetically engineered cell are integrated with the construct described in the first aspect of the present invention, or the genetically engineered cell contains the vector or vector combination described in the first aspect of the present invention.

[0108] Preferably, the genetically engineered cells include prokaryotic cells and eukaryotic cells.

[0109] Furthermore, the eukaryotic cells are selected from mammalian cells, plant cells, yeast cells, insect cells or any combination thereof.

[0110] Further, the yeast cell is selected from Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia aminuta, Ogataeaminuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia alictaria, Pichia g uercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia asp., Saccharomyces cerevisiae. cerevisiae), brewer's yeast, sugar cane molasses yeast, Saccharomyces sp., Hansenula polymorpha, Candida utilis, Kluyveromyces, or a combination thereof.

[0111] Furthermore, the Kluyveromyces further includes: Kluyveromyces lactis (Kluyveromyces, K.lactis), Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, or a combination thereof.

[0112] The fourth aspect of the present invention provides an in vitro protein synthesis system, characterized in that it comprises the construct described in the first aspect of the present invention, the vector or vector combination described in the second aspect of the present invention, or a cell extract prepared from the genetically engineered cell described in the third aspect of the present invention.

[0113] In another preferred embodiment, the eukaryotic in vitro biosynthesis system includes a eukaryotic in vitro protein synthesis system.

[0114] In another preferred embodiment, the eukaryotic in vitro biosynthesis system is selected from one of the following groups: 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.

[0115] In another preferred embodiment, the eukaryotic in vitro protein synthesis system is selected from one of the following groups: 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.

[0116] In another preferred embodiment, the yeast in vitro biosynthesis system (such as a yeast in vitro protein synthesis system) is a Kluyveromyces in vitro biosynthesis system (such as a Kluyveromyces in vitro protein synthesis system) (preferably a Kluyveromyces lactis or a Kluyveromyces marxianus in vitro biosynthesis system) (such as a Kluyveromyces lactis or a Kluyveromyces marxianus in vitro protein synthesis system).

[0117] The fifth aspect of the present invention provides the use of the construct according to the first aspect of the present invention, the vector or vector combination according to the second aspect of the present invention, the genetically engineered cell according to the third aspect of the present invention, or the in vitro protein synthesis system according to the fourth aspect of the present invention in in vitro protein synthesis.

[0118] The sixth aspect of the present invention provides a method for in vitro high-throughput synthesis of exogenous proteins, comprising the steps of:

[0119] (i) providing the nucleic acid construct according to the first aspect of the present invention in the presence of a eukaryotic in vitro biosynthesis system;

[0120] (ii) incubating the eukaryotic in vitro biosynthesis system of step (i) under suitable conditions for a period of time T1, thereby synthesizing the exogenous protein.

[0121] In another preferred embodiment, the method further comprises: (iii) optionally isolating or detecting the exogenous protein from the eukaryotic in vitro biosynthesis system.

[0122] In another preferred embodiment, the eukaryotic in vitro biosynthesis system is a yeast in vitro biosynthesis system (such as a yeast in vitro protein synthesis system).

[0123] In another preferred embodiment, the yeast in vitro biosynthesis system (such as a yeast in vitro protein synthesis system) is a Kluyveromyces in vitro biosynthesis system (such as a Kluyveromyces in vitro protein synthesis system) (preferably a Kluyveromyces lactis in vitro biosynthesis system) (such as a Kluyveromyces lactis in vitro protein synthesis system).

[0124] In another preferred embodiment, in step (ii), the reaction temperature is 20-37°C, preferably 22-35°C.

[0125] In another preferred embodiment, in the step (ii), the reaction time is 1-10 h, preferably, 2-8 h.

[0126] The present invention enhances the yield of cell-free expression protein by optimizing 3-UTR, terminator sequence, signal peptide sequence, kozak sequence, IRES sequence, Ω sequence and promoter sequence, saves time and capital costs, and greatly promotes the commercial application of protein in scientific research, medicine, health, beauty, food and other fields.

[0127] The main advantages of the present invention include:

[0128] (1) The present invention uses the optimized 3-UTR and the coding sequence of the foreign protein and optionally the signal peptide SP as a nucleic acid construct, which is applied to the in vitro protein synthesis system of the present invention, and can be used to improve the expression and purification of the foreign protein.

[0129] (2) Compared with other cells, Kluyveromyces lactis can be used in the production of proteins in the fields of food, medicine, skin care and beauty due to its safety and efficiency. In addition, the in vitro protein synthesis system has advantages such as adaptability to high-throughput protein synthesis screening, short protein synthesis time and low cost. Therefore, the in vitro protein synthesis system derived from Kluyveromyces lactis cells can also be widely used in related fields.

[0130] (3) The present invention also optimizes the IRES, Ω sequence, terminator and other sequences based on the basic sequence to further improve the expression and purification effects of the target exogenous protein, and more importantly, it can increase the possibility of synthesizing different proteins in the in vitro protein synthesis system of Kluyveromyces lactis. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 It is a schematic diagram of the DNA elements of D2P. T7P is the T7 promoter sequence, and T7T is the T7 terminator sequence.

[0132] Figure 2 It is a plasmid PCR gel image. M is a marker, and 1 to 4 are PCR products of plasmids SP517, SP533, SP506, and 1.08t bacterial solution, respectively. SP517 is a plasmid in which the SP basic sequence in the 1.08t plasmid is replaced with the sequence shown in SEQ ID NO.85; SP533 is a plasmid in which the SP basic sequence in the 1.08t plasmid is replaced with the sequence shown in SEQ ID NO.86; SP546 is a plasmid in which the SP basic sequence in the 1.08t plasmid is replaced with the sequence shown in SEQ ID NO.84. DETAILED DESCRIPTION

[0133] The advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0134] The present invention optimizes the 3-UTR sequence, enhances the yield of cell-free expression protein, saves time and financial costs, and greatly promotes the commercial application of protein in scientific research, medicine, health, beauty, food and other fields.

[0135] Coding sequence of foreign protein (foreign DNA)

[0136] As used herein, the term "coding sequence of exogenous protein" and "exogenous DNA" are used interchangeably, both referring to exogenous DNA molecules used to guide protein synthesis. Typically, the DNA molecules are linear or circular. The DNA molecules contain sequences encoding exogenous proteins.

[0137] In the present invention, examples of sequences encoding foreign proteins include (but are not limited to): genomic sequences, cDNA sequences. The sequences encoding foreign proteins also include promoter sequences, 5' untranslated sequences, and 3' untranslated sequences.

[0138] In the present invention, the selection of the exogenous DNA is not particularly limited. Generally, the exogenous DNA is selected from one of the following groups: exogenous DNA encoding luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of the variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.

[0139] The exogenous DNA can also be selected from one of the following groups: exogenous DNA encoding α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.

[0140] In a preferred embodiment, the exogenous DNA encodes a protein selected from the following groups: 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.

[0141] Nucleic acid constructs

[0142] The first aspect of the present invention provides a nucleic acid construct, wherein the nucleic acid construct has a structure of formula I from 5' to 3':

[0143] Z1-Z2(I)

[0144] In the formula,

[0145] Z1 and Z2 are respectively elements used to constitute the structure;

[0146] Each "-" is independently a bond or a nucleotide linking sequence;

[0147] Z1 is the coding sequence of no or foreign protein;

[0148] Z2 is a nucleic acid sequence without or without a 3' untranslated region (3-UTR),

[0149] The Z2 and Z1 are modified sequences of the wild-type sequence;

[0150] The nucleic acid construct is capable of enhancing cell-free expression of a protein.

[0151] In a preferred example, the nucleic acid construct has a structure of Formula I from 5' to 3':

[0152] Z3-Z1-Z2(I)

[0153] In the formula,

[0154] Z3, Z1, and Z2 are respectively elements used to constitute the structure;

[0155] Each "-" is independently a bond or a nucleotide linking sequence;

[0156] Z3 is the coding sequence of the signal peptide;

[0157] Z1 is the coding sequence of no or foreign protein;

[0158] Z2 is the nucleic acid sequence of the 3' untranslated region (3-UTR),

[0159] The Z3 and Z2 are modified sequences of the wild-type sequence;

[0160] The nucleic acid construct is capable of enhancing cell-free expression of a protein.

[0161] The Z3 is a coding sequence of a signal peptide, and the source is selected from the group consisting of one or more cells selected from the group consisting of prokaryotic cells and eukaryotic cells. In another preferred embodiment, the signal peptide source is selected from the group consisting of one or more cells selected from the group consisting of human, Chinese hamster ovary cells (CHO), insect cells, wheat germ cells, rabbit reticulocytes, and yeast cells.

[0162] The nucleic acid construct further comprises a coding sequence of a signal peptide, wherein the amino acid sequence of the signal peptide is selected from one of the following groups:

[0163] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.72;

[0164] In another preferred embodiment, the coding sequence of the signal peptide is selected from one of the following groups;

[0165] (a) a polynucleotide represented by any one of SEQ ID NO.73 to SEQ ID NO.144;

[0166] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.73 to SEQ ID NO.144;

[0167] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 73 to SEQ ID NO. 144;

[0168] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0169] In a preferred embodiment, the coding sequence of the signal peptide is selected from the following group:

[0170] (a) nucleotides encoding the polypeptides shown in SEQ ID NO.1 to SEQ ID NO.72;

[0171] (b) a polynucleotide as shown in any one of SEQ ID NO.73 to SEQ ID NO.144 in the table;

[0172] (c) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.73 to SEQ ID NO.144 shown in Table 1;

[0173] (d) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 73 to SEQ ID NO. 144;

[0174] (e) A polynucleotide complementary to the polynucleotide described in any one of (a) to (d).

[0175]

[0176]

[0177]

[0178]

[0179] Furthermore, the coding sequence of the exogenous protein represented by Z1 comes from prokaryotes or eukaryotes.

[0180] In another preferred embodiment, the coding sequence of the exogenous protein comes from animals, plants, or pathogens.

[0181] In another preferred embodiment, the coding sequence of the exogenous protein is from mammals, preferably primates, rodents, including humans, mice, and rats.

[0182] In another preferred embodiment, the coding sequence of the exogenous protein is selected from one of the following groups: exogenous DNA encoding luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, antibody variable region, luciferase mutant DNA or a combination thereof.

[0183] In another preferred embodiment, the exogenous protein is selected from one of the following groups: luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.

[0184] In another preferred embodiment, the coding sequence of the 3' untranslated region (3-UTR) is selected from one of the following groups:

[0185] (a) a polynucleotide shown in any one of SEQ ID NO.145 to SEQ ID NO.171 in Table 2;

[0186] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.145 to SEQ ID NO.171;

[0187] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 145 to SEQ ID NO. 171;

[0188] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0189]

[0190]

[0191]

[0192] Furthermore, the nucleic acid construct further comprises an IRES element, a kozak sequence, an Ω sequence, or a combination thereof.

[0193] Furthermore, the IRES element is derived from one or more cells selected from the following groups: human, Chinese hamster ovary cell (CHO), insect cell, wheat germ cell, rabbit reticulocyte.

[0194] In another preferred embodiment, the IRES element is selected from one of the following groups:

[0195] (a) a polynucleotide as shown in any one of SEQ ID NO.172 to SEQ ID NO.248;

[0196] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.172 to SEQ ID NO.248;

[0197] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 172 to SEQ ID NO. 248;

[0198] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0199]

[0200]

[0201]

[0202]

[0203] In another preferred embodiment, the kozak sequence is selected from one of the following groups:

[0204] (a) a polynucleotide as shown in any one of SEQ ID NO.249 to SEQ ID NO.254 in Table 4;

[0205] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to the sequence shown in any one of SEQ ID NO.249 to SEQ ID NO.254;

[0206] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 249 to SEQ ID NO. 254;

[0207] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0208]

[0209] In another preferred embodiment, the Ω sequence is selected from one of the following groups:

[0210] (a) a polynucleotide as shown in any one of SEQ ID NO.255 ​​to SEQ ID NO.269;

[0211] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.255 ​​to SEQ ID NO.269;

[0212] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 255 to SEQ ID NO. 269;

[0213] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0214]

[0215] In another preferred embodiment, the IRES element, the kozak sequence and the Ω sequence are combined to form a 5' untranslated region (5-UTR).

[0216] In another preferred embodiment, the 5' untranslated region (5-UTR) is selected from one of the following groups:

[0217] (a) a polynucleotide as shown in any one of SEQ ID NO.270 to SEQ ID NO.298 in Table 6;

[0218] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.270 to SEQ ID NO.298;

[0219] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 270 to SEQ ID NO. 298;

[0220] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0221]

[0222]

[0223]

[0224] In another preferred embodiment, the nucleic acid construct has a structure of formula II from 5' to 3': Y1-Y2-Y3-Z3-Z1-Z2 (I)

[0225] In the formula,

[0226] Y1, Z3, Z1, and Z2 are respectively elements used to constitute the construct;

[0227] Each "-" is independently a bond or a nucleotide linking sequence;

[0228] Z3 is the coding sequence of the signal peptide;

[0229] Z1 is the coding sequence of no or foreign protein;

[0230] Z2 is the nucleic acid sequence of the 3' untranslated region (3-UTR);

[0231] Y1 is absent or is an enhancer element, preferably an IRES element;

[0232] Y2 is none or a Kozak sequence;

[0233] Y3 is None or an Ω sequence.

[0234] Furthermore, the 5' end upstream of the nucleic acid construct also includes a promoter.

[0235] In another preferred embodiment, the promoter is selected from one of the following groups: T7 promoter, T3 promoter, SP6 promoter, or a combination thereof. More preferably, the promoter is T7 promoter (SEQ ID NO. 299: taatacgactcactatagg).

[0236] Furthermore, the 3' end downstream of the nucleic acid construct also includes a terminator.

[0237] In another preferred embodiment, the terminator is selected from one of the following groups: T7 terminator, T3 terminator, SP6 terminator, or a combination thereof.

[0238] In another preferred embodiment, the terminator sequence is selected from one of the following groups:

[0239] (a) encoding a polynucleotide as shown in any one of SEQ ID NO.300 to SEQ ID NO.304;

[0240] (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.300 to SEQ ID NO.304;

[0241] (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 300 to SEQ ID NO. 304;

[0242] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0243] Table 7 Terminator sequence table (DNA sequence)

[0244]

[0245] In another preferred embodiment, the nucleic acid construct has a structure of Formula II from 5' to 3':

[0246] X1-Y1-Y2-Y3-Z3-Z1-Z2-X2(I)

[0247] In the formula,

[0248] Y1, Z3, Z1, and Z2 are respectively elements used to constitute the construct;

[0249] Each "-" is independently a bond or a nucleotide linking sequence;

[0250] Z3 is the coding sequence of the signal peptide;

[0251] Z1 is the coding sequence of no or foreign protein;

[0252] Z2 is the nucleic acid sequence of the 3' untranslated region (3-UTR);

[0253] Y1 is absent or is an enhancer element, preferably an IRES element;

[0254] Y2 is none or a Kozak sequence;

[0255] Y3 is none or an Ω sequence;

[0256] X1 is the none or promoter series;

[0257] X2 is none or the terminator series.

[0258] In another preferred embodiment, the construct further comprises an element or a combination thereof selected from the following groups: a promoter, a terminator, a poly(A) element, a transport element, a gene targeting element, a selection marker gene, an enhancer, a resistance gene, and a transposase encoding gene.

[0259] A variety of selective marker genes can be applied to the present invention, including but not limited to: nutritional deficiency markers, resistance markers, and reporter gene markers. The application of selective markers plays a role in the screening of recombinant cells (recombinants), so that the recipient cells can be significantly distinguished from untransformed cells. Nutritional deficiency markers are complementary to the mutant genes of the recipient cells through the transferred marker genes, so that the recipient cells show wild-type growth. Resistance markers refer to the transfer of resistance genes into recipient cells, and the transferred genes make the recipient cells show drug resistance under certain drug concentrations. As a preferred embodiment of the present invention, resistance markers are used to achieve convenient screening of recombinant cells.

[0260] In the present invention, the nucleic acid construct of the present invention is applied in the in vitro protein synthesis system of the present invention, and the biotin-binding technology is used after the reaction is completed, so that the expression and purification of the exogenous protein can be completed. Specifically, the relative light unit value of the amount of enhanced green fluorescent protein synthesized by using the nucleic acid construct of the present invention is relatively high.

[0261] Carrier

[0262] The present invention also provides a vector or vector combination, wherein the vector contains the nucleic acid construct of the present invention. Preferably, the vector is selected from: bacterial plasmid, bacteriophage, yeast plasmid, or animal cell vector, shuttle vector; the vector is a transposon vector. Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used.

[0263] Genetically engineered cells

[0264] Those skilled in the art can use well-known methods to construct expression vectors containing the promoter and / or target gene sequence of the present invention, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

[0265] The present invention also provides a genetically engineered cell, wherein the genetically engineered cell contains the construct or vector combination, or the genetically engineered cell chromosome is integrated with the construct or vector. In another preferred embodiment, the genetically engineered cell further comprises a vector containing a transposase gene or the transposase gene is integrated into its chromosome.

[0266] Preferably, the genetically engineered cells are eukaryotic cells.

[0267] In another preferred embodiment, the eukaryotic cells include (but are not limited to): yeast cells (preferably, Kluyveromyces cells, more preferably, Kluyveromyces lactis cells).

[0268] 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, Agrobacterium; or lower eukaryotic cells, such as plant cells, yeast cells; or higher eukaryotic cells, such as insect cells, animal cells, etc. Those skilled in the art are aware of how to select appropriate vectors and genetically engineered cells. Transformation of genetically engineered cells with recombinant DNA can be carried out using 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 the CaCl2 method or electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Transformation of plants can also be carried out using methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, young embryo transformation method, flower bud soaking method, etc.

[0269] In vitro high-throughput protein synthesis system

[0270] In the present invention, the in vitro high-throughput protein synthesis system or "D2P" system includes but is not limited to IVTT reaction (in vitro transcription and translation reaction). In the present invention, IVTT reaction is preferred. IVTT reaction, corresponding to IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as D-to-P system, D_to_P system, DNAto-Protein system; the corresponding in vitro protein synthesis method is also called D2P method, D-to-P method, D_to_P method, DNA-to-Protein method, which has the same meaning as "in vitro cell-free protein synthesis system", "in vitro expression system", "in vitro protein synthesis system", "in vitro protein synthesis reaction system", "cell-free protein synthesis system" and other expressions. Protein in vitro synthesis system, in vitro protein synthesis system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system) and other descriptions. Including in vitro translation system, in vitro transcription and translation system (IVTT system), etc. We also call the in vitro protein synthesis system "Protein Synthesis Factory". In vitro protein synthesis reaction refers to the reaction of synthesizing protein in an in vitro cell-free synthesis system, which at least includes the translation process.

[0271] Protein synthesis system

[0272] In vitro protein synthesis systems generally refer to products that complete 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. At present, the commercial in vitro protein expression systems that are often tested include Escherichia coli extract (ECE), rabbit reticulocyte lysate (RRL), wheat germ extract (WGE), insect cell extract (ICE) and human cell systems. Compared with traditional in vivo recombinant expression systems, in vitro synthesis systems have many advantages, such as the ability to express special proteins that are toxic to cells or contain non-natural amino acids (such as D-amino acids), the ability to directly use PCR products as templates to simultaneously synthesize multiple proteins in parallel, and to carry out high-throughput drug screening and proteomics research.

[0273] Among them, yeast has the advantages of simple cultivation, efficient protein folding, and post-translational modification. Saccharomyces cerevisiae and Pichia pastoris are model organisms for expressing complex eukaryotic proteins and membrane proteins, and yeast can also be used as a raw material for preparing in vitro translation systems.

[0274] Kluyveromyces is a terminal ascospore yeast, of which Kluyveromyces marxianus and Kluyveromyces lactis are widely used in industry. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as super strong secretion ability, better large-scale fermentation characteristics, food safety level, and the ability to modify proteins after translation.

[0275] In the present invention, a preferred protein synthesis system is an in vitro protein synthesis system. In the present invention, the in vitro protein synthesis system is not particularly limited. A preferred in vitro protein synthesis system is a Kluyveromyces expression system (more preferably, a Kluyveromyces lactis expression system).

[0276] In the present invention, the in vitro protein synthesis system comprises:

[0277] (a) yeast cell extract;

[0278] (b) polyethylene glycol;

[0279] (c) optionally exogenous sucrose; and

[0280] (d) an optional solvent, wherein the solvent is water or an aqueous solvent.

[0281] In a particularly preferred embodiment, the in vitro protein synthesis system provided by the present invention comprises: yeast cell extract, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, RNase inhibitor, luciferin, luciferase DNA, and RNA polymerase.

[0282] In the present invention, the RNA polymerase is not particularly limited and can be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.

[0283] In the present invention, the proportion of the cell extract in the in vitro protein synthesis system is not particularly limited. Usually, the cell extract accounts for 20-70% of the in vitro protein synthesis system, preferably 30-60%, and more preferably 40-50%.

[0284] In the present invention, the cell extract does not contain intact cells, and a typical cell extract includes ribosomes, transfer RNA, aminoacyl tRNA synthetase, initiation factors and elongation factors required for protein synthesis, and termination release factors for protein translation. In addition, the cell extract also contains some other proteins derived from the cytoplasm of the cell, especially soluble proteins.

[0285] In the present invention, the protein content of the cell extract is 10-100 mg / mL, preferably 20-80 mg / mL. The method for determining the protein content is the Coomassie Brilliant Blue determination method.

[0286] In the present invention, the preparation method of the cell extract is not limited. A preferred preparation method comprises the following steps:

[0287] (i) providing cells;

[0288] (ii) washing the cells to obtain washed cells;

[0289] (iii) disrupting the washed cells to obtain a crude cell extract;

[0290] (iv) subjecting the crude cell extract to solid-liquid separation to obtain a liquid portion, namely the cell extract.

[0291] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0292] In a preferred embodiment, the centrifugation is performed in a liquid state.

[0293] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000 g, preferably 8000-30000 g.

[0294] In the present invention, the centrifugation time is not particularly limited, and a preferred centrifugation time is 0.5 min-2 h, preferably 20 min-50 min.

[0295] In the present invention, the centrifugation temperature is not particularly limited. Preferably, the centrifugation is performed at 1-10°C, more preferably, at 2-6°C.

[0296] In the present invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to use a washing liquid at a pH of 7-8 (preferably 7.4). The washing liquid is not particularly limited. Typically, the washing liquid is selected from one of the following groups: potassium 4-hydroxyethylpiperazineethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

[0297] In the present invention, the cell disruption treatment method is not particularly limited. A preferred cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen cryogenic) disruption.

[0298] The nucleoside triphosphate mixture in the in vitro protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate. In the present invention, the concentration of various mononucleotides is not particularly limited, and usually the concentration of each mononucleotide is 0.5-5mM, preferably 1.0-2.0mM.

[0299] The amino acid mixture in the in vitro protein synthesis system may include natural or non-natural amino acids, and may include D-type or L-type amino acids. Representative amino acids include (but are not limited to) 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 generally 0.01-0.5 mM, preferably 0.02-0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.

[0300] In a preferred embodiment, the in vitro protein synthesis system further contains polyethylene glycol or its analogs. The concentration of polyethylene glycol or its analogs is not particularly limited. Generally, 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 PEG 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 polyethylene glycols of various other molecular weights (such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).

[0301] In a preferred embodiment, the in vitro protein synthesis system further contains sucrose. The concentration of sucrose is not particularly limited, and generally, the concentration of sucrose is 0.03-40wt%, preferably 0.08-10wt%, and more preferably 0.1-5wt%, based on the total weight of the protein synthesis system.

[0302] A particularly preferred in vitro protein synthesis system (IVTT reaction solution) contains the following components in addition to cell extracts: 22 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg / mL creatine phosphate kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, 8-20 ng / μl exogenous protein DNA, and 0.027-0.054 mg / mL T7 RNA polymerase.

[0303] The present invention provides an in vitro high-throughput protein synthesis method, comprising the steps of:

[0304] (i) providing the nucleic acid construct according to the first aspect of the present invention in the presence of an in vitro protein synthesis system;

[0305] (ii) incubating the in vitro protein synthesis system of step (i) for a period of time T1 under suitable conditions, thereby synthesizing the exogenous protein.

[0306] In another preferred embodiment, the method further comprises: (iii) optionally isolating or detecting the exogenous protein from the in vitro protein synthesis system.

[0307] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not indicated in the following examples are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0308] Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products. In the examples of the present invention, the IVTT reaction solution used is: lactic acid Kluyveromyces cell extract, 22mM, pH 7.4 4-hydroxyethylpiperazineethanesulfonic acid, 30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, 1.5-4mM nucleoside triphosphate mixture, 0.08-0.24mM amino acid mixture, 25mM creatine phosphate, 1.7mM dithiothreitol, 0.27mg / mL creatine phosphate kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, 8-20ng / μl exogenous protein DNA, 0.027-0.054mg / mL T7RNA polymerase.

[0309] To verify the present invention, Figure 1 The plasmid 1.08t having the structure shown and containing the following basic sequence was used as a template for optimization experiments.

[0310]

[0311]

[0312] Example 1 Activity screening of gene synthesis

[0313] 1. Gene synthesis

[0314] IRES and terminator sequences were used for gene synthesis.

[0315] 2. Plasmid transformation

[0316] Add 1 μl of plasmid to 20 μl of DH5α, place on ice for 30 min, heat shock at 42°C for 45 s, place on ice for 2 min, add 500 μl of culture medium, and culture at 37°C with shaking for 1 h (200 rpm). Pipette 100 μl onto LB (containing antibody) plate, culture at 37°C upside down for 12-16 h, take out and store in a refrigerator at 4°C. Finally, select a single clone for sequencing to verify the sequence is correct.

[0317] 3. Plasmid extraction

[0318] (1) Collecting bacteria: Centrifuge the bacterial culture solution after 12 to 16 hours of culture at 4000 rpm for 10 min at 4°C, discard the supernatant, and after a short centrifugation, use a pipette to remove the residual liquid on the surface of the precipitate.

[0319] (2) Add 400 μl of P1 to the precipitate and vortex for more than 2 minutes to completely disperse the bacterial precipitate in P1.

[0320] (3) Add 500 μl of P2 to each portion and gently invert the 2 ml EP tube 8 times.

[0321] (4) Add 700 μl of P3 to each aliquot. Gently invert the 2 ml EP tube 8 times.

[0322] (5) Centrifuge at 12000 rpm for 15 min at 4°C to remove the precipitate

[0323] (6) Take the supernatant and mix it with 100 μl of pre-shaken magnetic beads. Incubate at room temperature for 5 min. Invert the EP tube every 1 min to ensure that the magnetic beads and supernatant are fully mixed.

[0324] (7) Use a magnetic stand to absorb the magnetic beads and pour out the supernatant. (Method: The magnetic absorption time is about 5-10 seconds. When the supernatant turns slightly yellow, pour out the supernatant and leave the magnetic stand in time to prevent the magnetic beads from binding too tightly and being unable to disperse in the next step. Since the conformation of the plasmid is easily destroyed by external forces, try to avoid vortexing during the washing process. The quality of the plasmid conformation affects the IVTT activity.)

[0325] (8) Wash the magnetic beads with 1 ml of PR. Since the magnetic beads are not tightly bound in the previous step, you can shake them loose by hand for 1 min. Use a magnetic rack to remove the supernatant, using the same method as above.

[0326] (9) Wash the magnetic beads with 1.2 ml of W. Since the magnetic beads are not tightly bound in the previous step, you can shake them loose by hand for 2 minutes. Use a magnetic rack to remove the supernatant, using the same method as above.

[0327] (10) Repeat step 9.

[0328] (11) Place the EP tube on the magnetic rack and use a hair dryer to dry the magnetic beads (at minimum power). After about 12 minutes, the completely dried magnetic beads should be cracked and there should be no water marks on the side of the magnetic beads close to the EP tube.

[0329] (12) Use the tip of a gun to gently scrape the dried magnetic beads to the bottom of the EP tube and add 400 μl of elution buffer. Vortex for 15 seconds and shake the EP tube intermittently within 5 minutes to ensure sufficient elution. Use a magnetic rack to collect the supernatant.

[0330] (13) Centrifuge at 12,000 rpm at 4°C for 5 min to remove residual magnetic beads.

[0331] (14) Take the supernatant with a pipette and centrifuge at 12,000 rpm for 10 min at 4°C to remove residual magnetic beads.

[0332] (15) Take the supernatant and detect the plasmid concentration.

[0333] (16) Agarose gel (1%) electrophoresis was performed to observe the plasmid morphology. The results are as follows: Figure 2 As shown, Figure 2 1 to 4 are PCR gel images of the corresponding plasmids SP517, SP533, SP546, and 1.08t bacterial solution, respectively, wherein SP517 is a plasmid in which the SP basic sequence in the 1.08t plasmid is replaced with the sequence shown in SEQ ID NO.85; SP533 is a plasmid in which the SP basic sequence in the 1.08t plasmid is replaced with the sequence shown in SEQ ID NO.86; SP506 is a plasmid in which the SP basic sequence in the 1.08t plasmid is replaced with the sequence shown in SEQ ID NO.84.

[0334] 4. Plasmid PCR

[0335] Pipette 1 μl of 100 ng / μl plasmid as template for plasmid PCR. The system and procedure used are as follows:

[0336]

[0337]

[0338]

[0339] Protein expression detection

[0340] Add 10 μl of PCR product to 300 μl IVTT, react at 30°C, 100 rpm for 3 h, and then detect the RFU value.

[0341] Activity optimization experiment: Use 1.08t bacterial liquid PCR product of the above basic sequence as a comparative example, detect its RFU value, and use it as the benchmark value RFU 0 , the RFU value of the test case RFU x RFU 0 As the activity value, the calculation formula of the activity value is as follows:

[0342] Activity value = RFU x / RFU 0 *100%

[0343] The higher the activity value, the better the optimization effect of the test case. On the contrary, the lower the activity value, the worse the optimization effect of the test case.

[0344] 1. 3-UTR optimization protein expression detection experimental results (Compared to 1.08t, it is just modified 3-UTR , that is, only 1.08t plasmid 3-UTR The base sequence is replaced by the following 3-UTR sequence)

[0345]

[0346]

[0347] 2. Results of the terminator optimization protein expression test (Compared to 1.08t, it is just modified Terminator , that is, only 1.08t plasmid Terminator The base sequence is replaced by the following Terminator sequence)

[0348] Experiment number Terminator sequence Activity value 46 SEQ ID NO.300 104％ 47 SEQ ID NO.301 101％ Comparative Example SEQ ID NO.312 100％

[0349] Example 2 Activity screening of random mutations

[0350] 1. Construction of plasmid

[0351] The signal peptide, partial kozak, IRES and Ω sequences were constructed by PCR.

[0352] 1.08t was used as a template and the corresponding primers were used for PCR. The PCR product was digested with DpnI in a 37°C incubator for at least 4 hours before transformation (DH5α).

[0353] The conditions and procedures for PCR construction are as follows:

[0354]

[0355]

[0356] 2. Transformation of mutant plasmids

[0357] Add 10 μl of the target PCR product after DpnI digestion to 100 μl of DH5α, place on ice for 30 min, heat shock at 42°C for 45 s, place on ice for 2 min, add 500 μl of culture medium, and shake culture at 37°C for 1 h (200 rpm). Pipette 100 μl onto LB (containing antibody) plate, invert and culture at 37°C for 12-16 h, and store in a 4°C refrigerator.

[0358] 3. Pick a single colony and shake it

[0359] Use a sterilized 10 μl white pipette tip to pick a single colony from the plate into a 96-well plate. Each well contains 80 μl of LB medium containing ampicillin resistance. Shake the plate at 37°C, 200 rpm overnight.

[0360] 4. Bacterial liquid PCR

[0361] 1 μl of the bacterial solution from the 96-well plate was taken as a template for bacterial solution PCR. The system and procedure used were as follows:

[0362]

[0363]

[0364] 5. Protein expression detection

[0365] Add 10 μl of bacterial solution PCR product to 300 μl IVTT, react at 30°C, 100 rpm for 3 h, and then detect the RFU value.

[0366] Activity optimization experiment: Use 1.08t bacterial liquid PCR product containing the above basic sequence as a comparative example, detect its RFU value, and use it as the benchmark value RFU 0 , the RFU value of the test case RFU x RFU 0As the activity value, the calculation formula of the activity value is as follows:

[0367] Activity value = RFU x / RFU 0 *100%

[0368] The higher the activity value, the better the optimization effect of the test case. On the contrary, the lower the activity value, the worse the optimization effect of the test case.

[0369] According to the above detection comparison scheme, the results of specific optimization or screening experiments are as follows:

[0370] one, Signal peptide (SP) optimization experiment (compared with 1.08t, only SP was optimized, that is, only SP in 1.08t plasmid was replaced with the following signal peptide coding sequence)

[0371] Experiment (I)

[0372] Experiment number Signal peptide coding sequence RFU Activity value 1 SEQ ID NO.88 6907 159％ 2 SEQ ID NO.85 6528 150％ 3 SEQ ID NO.92 6408 147％ 4 SEQ ID NO.87 6127 141％ 5 SEQ ID NO.93 6069 140％ 6 SEQ ID NO.86 6064 139％ 7 SEQ ID NO.91 6027 139％ 8 SEQ ID NO.89 5943 137％ 9 SEQ ID NO.90 5854 135％ 10 SEQ ID NO.84 5825 134％ Comparative Example SEQ ID NO.309 4350 100％

[0373] Experiment (II)

[0374] Experiment number Signal peptide coding sequence Activity value 11 SEQ ID NO.73 124％ 12 SEQ ID NO.74 123％ 13 SEQ ID NO.75 118％ 14 SEQ ID NO.76 128％ 15 SEQ ID NO.77 115％ 16 SEQ ID NO.78 112％ 17 SEQ ID NO.79 121％ 18 SEQ ID NO.80 129％ 19 SEQ ID NO.81 122％ 20 SEQ ID NO.82 113％ 21 SEQ ID NO.83 129％ Comparative Example SEQ ID NO.309 100％

[0375] two, Screening experiment of kozak sequence (compared with 1.08t, it is just a modification of kozak, that is, only the The Kozak base sequence is replaced with the following Kozak sequence)

[0376] Experiment number Kozak sequence Activity value 22 SEQ ID NO.251 95％ 23 SEQ ID NO.252 90％ 24 SEQ ID NO.253 91％ 25 SEQ ID NO.254 90％ Comparative Example SEQ ID NO.308 100％

[0377] three, IRES sequence screening experiment (compared with 1.08t, only the IRES in the 1.08t plasmid was modified The base sequence was replaced with the following IRES sequence)

[0378]

[0379] Four, Screening experiment of Ω sequence (compared with 1.08t, only the Ω sequence was modified, that is, only the Ω sequence in the 1.08t plasmid was The base sequence was replaced with the following IRES sequence)

[0380]

[0381]

[0382] It can be seen that the present invention has at least the following advantages:

[0383] (1) The present invention optimizes the IRES, Ω sequence, terminator and other sequences to further improve the expression and purification effects of the target exogenous protein, and more importantly, it can increase the possibility of synthesizing different proteins in the in vitro protein synthesis system of Kluyveromyces lactis.

[0384] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A nucleic acid construct having a structure of formula I from 5' to 3': Z1-Z2(I) In the formula, Z1 and Z2 are respectively elements used to constitute the structure; Each "-" is independently a bond or a nucleotide linking sequence; Z1 is the coding sequence of no or foreign protein; Z2 is the nucleic acid sequence of the 3' untranslated region (3-UTR), The Z2 and Z1 are modified sequences of the wild-type sequence; The nucleic acid construct is capable of enhancing cell-free expression of a protein.

2. The nucleic acid construct according to claim 3, wherein The connecting sequence indicated by "-" is a codon-optimized connecting sequence; preferably, the connecting sequence has a sequence that is not easy to form a secondary structure (such as an AT-rich sequence, no hairpin structure, no G-quadruplex, etc.), and is not rich in rare codons.

3. The nucleic acid construct according to claim 1, wherein The coding sequence of the exogenous protein shown in Z1 is from prokaryotes and eukaryotes; preferably, the coding sequence of the exogenous protein is from animals, plants, and pathogens; more preferably, the coding sequence of the exogenous protein is from mammals, preferably primates, rodents, including humans, mice, and rats; preferably, the exogenous protein is selected from natural proteins or mutant proteins or artificially designed proteins of soluble proteins, functional proteins, and active proteins; more preferably, the exogenous protein is selected from one of the following groups: luciferin protein, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutation, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody segment (scFV), thyroxine transporter, tyrosinase, xylanase, or a combination thereof.

4. The nucleic acid construct according to any one of claims 1 to 3, characterized in that: The coding sequence of the 3' untranslated region (3-UTR) shown in Z2 is selected from one of the following groups; (a) a polynucleotide as shown in any one of SEQ ID NO.145 to SEQ ID NO.171; (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.145 to SEQ ID NO.171; (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 145 to SEQ ID NO. 171; (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

5. The nucleic acid construct according to any one of claims 1 to 4, characterized in that: The nucleic acid construct further comprises a coding sequence of a signal peptide, wherein the amino acid sequence of the signal peptide is selected from one of the following groups: The amino acid sequences are shown as SEQ ID NO.1 to SEQ ID NO.

72.

6. The nucleic acid construct according to claim 5, characterized in that: The coding sequence of the signal peptide is selected from one of the following groups; (a) a polynucleotide represented by any one of SEQ ID NO.73 to SEQ ID NO.144; (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.73 to SEQ ID NO.144; (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 73 to SEQ ID NO. 144; (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

7. The nucleic acid construct according to any one of claims 1 to 6, characterized in that: The nucleic acid construct further comprises an enhancer element, a Kozak sequence, an Ω sequence, or a combination thereof.

8. The nucleic acid construct according to any one of claims 1 to 7, characterized in that: The IRES element is derived from one or more cells selected from the group consisting of: human cells, Chinese hamster ovary cells (CHO), insect cells, wheat germ cells, and rabbit reticulocytes; preferably, The IRES element is selected from one of the following groups; (a) a polynucleotide as shown in any one of SEQ ID NO.172 to SEQ ID NO.248; (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.172 to SEQ ID NO.248; (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 172 to SEQ ID NO. 248; (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

9. The nucleic acid construct according to claim 7 or 8, characterized in that: The kozak sequence is selected from one of the following groups: (a) a polynucleotide as shown in any one of SEQ ID NO.249 to SEQ ID NO.254; (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to the sequence shown in any one of SEQ ID NO.249 to SEQ ID NO.254; (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 249 to SEQ ID NO. 254; (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

10. The nucleic acid construct according to any one of claims 7 to 9, characterized in that: The Ω sequence is selected from one of the following groups: The Ω sequence is selected from one of the following groups; (a) a polynucleotide as shown in any one of SEQ ID NO.255 ​​to SEQ ID NO.269; (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.255 ​​to SEQ ID NO.269; (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 255 to SEQ ID NO. 269; (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

11. The nucleic acid construct according to any one of claims 7 to 10, characterized in that: The IRES element, kozak sequence and Ω sequence are combined to form a 5' untranslated region (5-UTR); Preferably, in another preferred embodiment, the 5' untranslated region (5-UTR) is selected from one of the following groups; (a) a polynucleotide as shown in any one of SEQ ID NO.270 to SEQ ID NO.298; (b) a polynucleotide having a nucleotide sequence homology of ≥30%, preferably 75% (preferably ≥85%, more preferably ≥90% or ≥95% or ≥98% or ≥99%) to any one of SEQ ID NO.270 to SEQ ID NO.298; (c) a polynucleotide having 1 to 20, preferably 1 to 10 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide represented by any one of SEQ ID NO. 270 to SEQ ID NO. 298; (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

12. A vector or a vector combination, wherein the vector or the vector combination comprises the nucleic acid construct according to any one of claims 1 to 11.

13. A genetically engineered cell, wherein the nucleic acid construct according to any one of claims 1 to 11 is integrated into one or more sites of the genome of the genetically engineered cell, or the genetically engineered cell contains the vector or vector combination according to claim 12; preferably, the genetically engineered cell comprises a prokaryotic cell or a eukaryotic cell, and more preferably, the genetically engineered cell is a yeast cell.

14. An in vitro protein synthesis system, characterized in that: A method comprising the nucleic acid construct according to any one of claims 1 to 11, the vector or vector combination according to claim 12, or a cell extract prepared from the genetically engineered cell according to claim 13.

15. Use of the nucleic acid construct according to any one of claims 1 to 11, the vector or vector combination according to claim 12, the genetically engineered cell according to claim 13 or the kit according to claim 14 in in vitro protein synthesis.

16. A method for in vitro cell-free protein synthesis, characterized in that: The in vitro high-throughput exogenous protein synthesis method comprises the steps of: (i) providing a nucleic acid construct according to any one of claims 1 to 11 as a synthesis template in the presence of a eukaryotic in vitro biosynthesis system; (ii) incubating the eukaryotic in vitro biosynthesis system of step (i) under suitable conditions for a period of time T1, thereby synthesizing the exogenous protein; preferably, in step (ii), the reaction temperature is 20-37°C, preferably, 20-25°C; preferably, in step (ii), the reaction time is 1-72h, preferably, 2-23h.

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