WO2024119434A1

WO2024119434A1 - Solubilization-assisting short peptide tag with acidic surface for improving expression efficiency of recombinant protein

Info

Publication number: WO2024119434A1
Application number: PCT/CN2022/137539
Authority: WO
Inventors: 赵晟; 李鹏; 孙秀莲; 仲明; 王志民
Original assignee: 宜明（济南）生物科技有限公司
Priority date: 2022-12-08
Filing date: 2022-12-08
Publication date: 2024-06-13
Also published as: WO2024119434A9

Abstract

Provided is an isolated peptide having an amino acid sequence of VDNKFNKEQQX1AFYEILHLPNLNEEQRNAFIQX2LKDDPX3X4SX5X6X7LX8EAX9X 10LNDAQPK (SEQ ID NO: 9), wherein X1-X10 are all negatively charged amino acids, preferably E or D. Further provided are a fusion protein comprising the peptide, a polynucleotide encoding the peptide or fusion protein, a construct and host cell comprising the polynucleotide, and a method for producing the fusion protein or the polypeptide of interest.

Description

An acidic surface-soluble short peptide tag for improving the expression efficiency of recombinant proteins

Technical Field

The invention belongs to the field of protein purification, and in particular relates to an acidic surface solubilizing short peptide label designed by computer-aided calculation.

Background technique

In recent years, with the continuous development of biotechnology, recombinant protein expression and purification technology has been valued and widely used. Among them, prokaryotic expression has the lowest cost, is the easiest to operate and has the highest purification efficiency. Escherichia coli (E. coli) is one of the most commonly used host organisms for recombinant protein production.

Many recombinant proteins rich in hydrophobic amino acid residues, especially those from non-prokaryotes, often face the problem of being difficult to dissolve and entering inclusion bodies in large quantities due to the lack of corresponding protein folding and protection mechanisms in prokaryotic cells. These recombinant proteins that precipitate during expression are largely inactivated even after re-dissolving.

In order to increase the expression amount, improve the correct conformation ratio and solubility of the product, and facilitate downstream purification operations, people use the method of fusion expression of solubilizing proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP), glutathione S-transferase (GST), etc. However, due to the large molecular weight of these solubilizing proteins themselves, there are still defects such as long folding time, limited solubilizing effect and easy interference with the activity of recombinant proteins. Therefore, the art needs to design and develop new solubilizing peptide molecules to assist the production and purification of recombinant proteins.

Summary of the invention

Since most proteins have acidic or neutral isoelectric points [Link A J et al., Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12, ELECTROPHORESIS, 1997, 18(8):1259-1313], the present invention uses computer-aided calculation to perform surface design of charge distribution on short stable protein domains, so that the surface contains multiple negative charges, thereby increasing the polarity of the domains themselves and making them more soluble in water, and being able to repel proteins with acidic isoelectric points, thereby reducing aggregation during protein purification, which not only plays an excellent solubilizing role, but also reduces interference with the folding process of the recombinant protein itself. The solvent-exposed surfaces of some bacterial receptor proteins, especially those containing helical bundle structures, have been shown to be very stable [Alexander P et al., Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures, Biochemistry, 1992. 31(14): 3597-3603]. These stable solvent-exposed surface regions can be used to design stable solubilizing peptides.

The acidic surface solubilizing peptide tag (Sacid) of the present invention is based on the B domain of Staphylococcus protein A [

N et al., A synthetic IgG-binding domain based on staphylococcal protein A, Protein Eng, 1(2): 107-113, 1987]. This domain is short and stable, containing only 57 amino acid residues and a molecular weight of only 8kD, which is suitable for charge distribution modification and addition to recombinant proteins as fusion tags. It can effectively solubilize recombinant proteins, stabilize the conformation of recombinant proteins during purification, improve the expression efficiency of recombinant proteins, and maintain their biological functional activity. The present invention grafts this modified short polypeptide with a surface rich in negative charge onto the target recombinant protein, which is more advantageous than the positively charged solubilizing tags published by other researchers in the early stage in terms of improving the expression efficiency, solubility and biological activity of recombinant proteins.

In _a first aspect, the _present invention provides an isolated _peptide comprising the amino acid sequence VDNKFNKEQQX1AFYEILHLPNLNEEQRNAFIQX2LKDDPX3X4SX5X6X7LX8EAX9X10LNDAQPK ₍ SEQ ID NO: ₉ ), wherein: _X1 _- _X10 are _all negatively _charged amino acids, preferably glutamic acid (E) or aspartic acid ( _D ₎ .

In a second aspect, the present invention provides an isolated polynucleotide encoding the peptide of the first aspect.

In a third aspect, the present invention provides an isolated fusion protein comprising a first peptide and a second peptide, wherein the first peptide is the peptide of the first aspect and the second peptide is a polypeptide of interest.

In a fourth aspect, the present invention provides an isolated polynucleotide encoding the fusion protein of the third aspect.

In a fifth aspect, the present invention provides a construct comprising the polynucleotide of the fourth aspect.

In a sixth aspect, the present invention provides a host cell comprising the polynucleotide of the fourth aspect or the construct of the fifth aspect, wherein the host cell is capable of expressing the fusion protein.

In a seventh aspect, the present invention provides a method for producing a fusion protein, comprising: (a) culturing the host cell of the sixth aspect under conditions suitable for expression of the fusion protein; and (b) recovering the fusion protein, optionally, (c) cleaving the fusion protein to release a polypeptide of interest and (d) recovering the polypeptide of interest.

In an eighth aspect, the present invention provides use of the peptide of the first aspect or the polynucleotide of the second aspect for producing a target protein.

In the ninth aspect, the present invention provides a method for producing a target protein, comprising: (a) expressing a fusion protein formed by fusion of the target protein and the peptide of the first aspect in a host cell; (b) cutting the fusion protein to release the target protein; and (c) optionally, isolating and/or purifying the target protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Sequence alignment results of amino acid sequences of different short peptide tags. Among them, Wt corresponds to SEQ ID NO: 4; Zbasic corresponds to SEQ ID NO: 7; Sacid corresponds to SEQ ID NO: 1; Sacid1 corresponds to SEQ ID NO: 2; and Sacid2 corresponds to SEQ ID NO: 3.

Figure 2: Electrostatic potential model of different short peptide tags at physiological pH; Figure A is the electrostatic potential model of the N-terminus (left) and C-terminus (right) of the B domain of Staphylococcal protein A at physiological pH; Figure B is the electrostatic potential model of the N-terminus (left) and C-terminus (right) of Zbasic at physiological pH; Figure C is the electrostatic potential model of the N-terminus (left) and C-terminus (right) of Sacid at physiological pH; Figure D is the electrostatic potential model of the N-terminus (left) and C-terminus (right) of Sacid 1 at physiological pH; Figure E is the electrostatic potential model of the N-terminus (left) and C-terminus (right) of Sacid 2 at physiological pH.

Figure 3: Solubility and isoelectric point predicted by computer-aided prediction (URL: https://protein-sol.manchester.ac.uk/). It predicts protein solubility based on sequence. Based on the observation of a bimodal distribution of solubility of cell-free expressed Escherichia coli proteins [Niwa T et al., Bimodal protein solubility distribution revealed by an aggregation analysis of the entire ensemble of Escherichia coli proteins, Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(11): 4201-4206], these measurements report the ratio of the soluble fraction of a protein (in the supernatant after centrifugation) to the total amount of the protein, rather than a thermodynamic property. The tool calculates 35 features, including: 20 amino acid compositions; 7 amino acid combinations: K-R, D-E, K+R, D+E, K+R-D-E, K+R+D+E, F+W+Y; 8 predicted features: length, isoelectric point, hydrophobicity, net charge of protein at pH=7.0, folding tendency, disordered region, sequence entropy, and beta chain tendency. A linear model combining 35 features gives a preliminary fit to the solubility data. The return value is between 0-1. If it is higher than 0.45 (the average value of the E. coli expressed protein data set Niwa et al (2009)), it means that the solubility of the protein may be higher than the average solubility of the E. coli expressed protein.

Figures A and B show the solubility of different solubility-enhancing tags. As can be seen from the figure, although the self-solubility of the acidic short peptide solubility tag (Sacid) of the present invention and the wild-type (WT) tag before modification is not much different, and is even slightly lower than the SUMO tag (Figure A), because its charged groups are concentrated on the surface, after fusion with the recombinant protein, it can interact with the positively charged protons in the water more effectively than other tags to form a hydration film, thereby helping the recombinant protein to dissolve. Therefore, after the Sacid tag is fused with the target protein, the solubility of the target protein can be significantly improved (Figure B). The acidic short peptide solubility tag (Sacid) of the present invention has a higher self-solubility than the known published positively charged tag (Zbasic) (Figure A). Both charges are distributed on the surface, but because the negative charge on the acidic surface can repel each other with more negative charges on the surface of most weakly acidic recombinant proteins, it is not easy to aggregate, and the solubility effect is better than that of the alkaline solubility peptide tag. Therefore, after fusion with the target protein, the solubility effect is significantly better than the positively charged tag (Zbasic) (Figure B). Similarly, compared with other commonly known solubilizing tags, the acidic short peptide solubilizing tag (Sacid) of the present invention also has a significantly better solubilizing effect.

Figures C and D show the isoelectric points of different solubilizing tags. It can be seen from the figure that the acidic short peptide solubilizing tag of the present invention exhibits better acidity. After fusion with the target protein, the pI of the fusion protein still exhibits a strong acidity.

Figure E shows the predicted characteristics of the acidic short peptide solubility tag (Sacid) of the present invention (net charge of the protein at pH = 7.0, folding tendency, etc.).

FIG4 is a schematic diagram of the protein expression and purification technology used in the present invention.

Figure 5: Electrophoretic identification of target proteins purified using different purification tags.

Figure A shows the situation when NsiI protein is expressed alone. M: protein molecular weight standard; F: Strep-tag II purification flow-through; W1, W2: Strep-tag II purification wash solution; E: Strep-tag II purification eluate. The arrow points to the target protein. It can be seen from the figure that when NsiI protein is expressed alone, its expression level is not high because it is prone to aggregation and precipitation before the protein is correctly folded. After purification, the target protein is significantly reduced or even completely lost.

Figure B shows the soluble expression of NeonGreen-NsiI fusion protein using NeonGreen as a solubilizing peptide to express NsiI. M: protein molecular weight standard; F: Strep-tag II purification flow-through; W1, W2: Strep-tag II purification wash solution; E: Strep-tag II purification eluate. The arrow points to the fusion protein, indicating that although the use of NeonGreen as a solubilizing protein fusion expression method can also achieve the purification and enrichment of the fusion protein, due to its large molecular weight, it still has defects such as long folding time, limited solubilization effect and easy interference with the activity of the recombinant protein. It can be seen from the figure that most of the protein exists in insoluble inclusion bodies and the fusion protein is easily degraded.

Figure C shows the electrophoresis identification of the fusion protein purified by Strep-tag II affinity column chromatography using a positively charged solubilizing tag (Zbasic) published by other researchers as a purification tag. M: protein molecular weight standard; F: Strep-tag II purification flow-through; W1, W2: Strep-tag II purification wash solution; E: Strep-tag II purification eluate. The arrow points to the fusion protein. Compared with Figure 5D, the published positively charged solubilizing tag (Zbasic) has a lower yield; this indirectly shows that the Sacid tag of the present invention can efficiently solubilize the recombinant protein, stabilize the conformation of the recombinant protein during the purification process, and improve the expression efficiency of the recombinant protein.

Figure D shows the electrophoresis identification of the fusion protein purified by Strep-tag II affinity column chromatography using Sacid as the purification tag using the technology of the present invention. M: protein molecular weight standard; F: Strep-tag II purification flow-through; W1: Strep-tag II purification wash solution; E: Strep-tag II purification eluate. The arrow points to the fusion protein. Compared with Figure 5C, the Sacid tag significantly improves the solubility of the fusion protein, and the target protein is obtained after Strep-tag II purification.

Figure 6: Comparison of purification of target protein using Sacid and NeonGreen purification tags

Figure A is the SDS PAGE of TEVP protein expressed using Sacid and NeonGreen as solubilizing peptides. M: protein molecular weight standard; ST: Sacid-TEVP fusion protein; NT: NeonGreen-TEVP fusion protein. The arrow points to the fusion protein. From the figure, only a thick band at 39KD of Sacid-TEVP can be observed, while the target band cannot be clearly seen when NeonGreen is used as a solubilizing protein fusion expression method.

Figure B is a Western blot of TEVP expressed using Sacid and NeonGreen as solubilizing peptides. M: protein molecular weight standard; ST: Sacid-TEVP fusion protein; NT: NeonGreen-TEVP fusion protein. The arrow points to the fusion protein. It can be seen from the figure that when Sacid is used as a solubilizing peptide, the Western blot results show a clear band at 39KD, but when NeonGreen is used as a solubilizing peptide, no target band is observed in the Western blot, which is basically consistent with the SDS PAGE result of Figure A. It shows that after the fusion expression of the present invention, the expression amount of the target protein has been greatly improved; compared with other tags, the Sacid tag of the present invention significantly improves the solubility of the fusion protein.

Figure 7: Activity test of purified protein, using a plasmid containing a single NsiI restriction site and a single HindIII restriction site as a substrate, after double restriction digestion reaction, 3392bp and 1987bp target bands were obtained. The purified NsiI enzyme was diluted in a gradient (10 ¹ to 10 ⁸ times) and then subjected to double restriction digestion reaction (37°C, 1 hour). Figure A shows the double restriction digestion identification of the NsiI protein expressed alone and the NeonGreen-NsiI protein; Figure B shows the double restriction digestion identification of the NsiI protein expressed by the positively charged solubilizing tag (Zbasic) and the Sacid-NsiI protein of the present invention. As can be seen from the figure, the NsiI protein expressed alone is almost inactive; the activities of the NsiI protein expressed by the NeonGreen-NsiI fusion protein and the positively charged solubility tag (Zbasic) are basically the same; and by using the technology of the present invention with Sacid as the purification tag, the activity of the purified protein Sacid-NsiI is greatly improved, which is higher than the activity of the NsiI protein expressed by the NeonGreen-NsiI fusion protein and the positively charged solubility tag (Zbasic).

Figure 8: Specific activities of enzymes purified in different ways (one unit is defined as the amount of enzyme required to digest 1 μg of plasmid DNA containing a single NsiI restriction site in 50 μl of reaction buffer at 37°C for 1 hour).

Detailed ways

Unless otherwise specified or obvious from the context, the abbreviations used herein have their conventional meanings in the fields of chemistry and biology, and all technical and scientific terms used in the present invention have the same meanings as those generally understood by those skilled in the art. Experimental methods not specifically described in the present invention are carried out according to the specific methods in the book "Molecular Cloning Laboratory Guide" (4th edition) by J. Sambrook, or according to the instructions of relevant products. The biological reagents used in the present invention can be obtained from commercial channels unless otherwise specified. Those skilled in the art can make various changes, modifications and substitutions without departing from the spirit of the present invention.

Unless otherwise indicated, nucleic acid sequences are referred to herein in a 5' to 3' direction from left to right; amino acid sequences are referred to herein in a 5' to 3' direction from left (upstream) to right (downstream).

The present invention is based on the amino acid sequence shown in the B domain of staphylococcal protein A, uses computer-assisted calculations to transform the C-terminal domain of the B structure of natural staphylococcal protein A, introduces negatively charged amino acids, concentrates the negatively charged amino acids on one side, and designs an acidic surface solubilizing short peptide tag (Sacid).

The present invention uses computer-assisted calculations to perform surface design of charge distribution on short stable protein domains, so that the surface contains multiple negative charges, and designs several acidic short peptide tags, namely Sacid (SEQ ID NO: 1), Sacid1 (SEQ ID NO: 2), and Sacid2 (SEQ ID NO: 3). It is found that the charge of the Sacid tag is relatively concentrated, more acidic than other designed tags, and can repel proteins with low isoelectric points, which can reduce aggregation during protein purification and play an excellent solubilizing role. This tag is fused with the target protein, and it is found that the tag can efficiently solubilize the recombinant protein, stabilize the conformation of the recombinant protein during the purification process, improve the expression efficiency of the recombinant protein, and maintain its biological functional activity. The method fuses the acidic surface solubilizing short peptide tag to be studied with the target protein, and quickly purifies the target protein through affinity chromatography.

In one embodiment, _X1 is D. In one embodiment, _X2 is E. In one embodiment, _X3 is E. In one embodiment, _X4 is E. In one embodiment, _X5 is D. In one embodiment, _X6 is E. In one embodiment, _X7 is E. In one embodiment, _X8 is E. In one embodiment, _X9 is D. In one embodiment, _X10 is D.

The isolated peptide provided by the present invention is an acidic surface solubilizing short peptide capable of improving the expression efficiency of recombinant proteins. It is based on the amino acid sequence of the B domain of Staphylococcus protein A (SEQ ID NO: 4), introduces negatively charged amino acids and concentrates the negatively charged amino acids on the surface. The protein solubility is predicted based on the sequence by computer-assisted prediction (see https://protein-sol.manchester.ac.uk/). The tool calculates 35 features, including: 20 amino acid compositions; 7 amino acid combinations: K-R, D-E, K+R, D+E, K+R-D-E, K+R+D+E, F+W+Y; 8 predicted features: length, isoelectric point, hydrophobicity, net charge of protein at pH=7.0, folding tendency, disordered region, sequence entropy, and β-chain tendency. The linear model combining the 35 features gives a preliminary fit to the solubility data. The predicted return value is between 0 and 1. If it is higher than 0.45 (the average value of the E. coli expressed protein dataset Niwa et al (2009)), it means that the solubility of the protein may be higher than the average solubility of the E. coli expressed protein.

Thus, in a preferred embodiment, the predicted solubility of the peptide shown in SEQ ID NO: 9 is greater than 0.45, as predicted based on https://protein-sol.manchester.ac.uk/. In one embodiment, the predicted solubility of the peptide shown in SEQ ID NO: 9 is equal to or greater than 0.70, as predicted based on https://protein-sol.manchester.ac.uk/.

In one embodiment, the isoelectric point pI of the soluble peptide of SEQ ID NO: 9 is equal to or less than 5.0.

In one embodiment, the peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and are defined as biological molecules composed of amino acid residues linked by peptide bonds.

In a second aspect, the present invention relates to a polynucleotide encoding the peptide of the first aspect.

As used herein, the terms "nucleotide sequence", "polynucleotide", "nucleic acid" and "nucleic acid sequence" are used interchangeably and refer to a macromolecule composed of multiple nucleotides connected by 3'-5'-phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequence of the polynucleotide of the present invention can be codon-optimized for different host cells (such as Escherichia coli) to improve the expression of the fusion protein. Methods for codon optimization are known in the art.

In a third aspect, the present invention relates to an isolated fusion protein comprising a first peptide and a second peptide, wherein the first peptide is a peptide according to the first aspect of the present invention and the second peptide is a polypeptide of interest.

As used herein, "target polypeptide" refers to any polypeptide or protein that can be produced and purified by the method of the present invention, non-limiting examples of which include enzymes, hormones, immunoglobulin chains, therapeutic polypeptides such as anti-cancer polypeptides, diagnostic polypeptides, or polypeptides that can be used for immunization purposes or biologically active fragments thereof, etc. The target polypeptide can be derived from any source, including microbial-derived polypeptides, mammalian-derived polypeptides, and artificial proteins (e.g., fusion proteins or mutant proteins), etc.

The target polypeptide can be a polypeptide or protein of any length. In one embodiment, the target polypeptide that can be produced and purified by the method of the present invention can have a length of 20-500 amino acid residues, for example, about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 amino acid residues.

In one embodiment, the polypeptide of interest described herein has an acidic, neutral or weakly basic isoelectric point lower than, equal to or slightly greater than 7.0, such as equal to or lower than 8.0, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0.

In some embodiments, the first peptide can be located upstream (N-terminal) or downstream (C-terminal) of the second peptide, preferably located upstream (N-terminal). In some embodiments, the first peptide is located upstream of the second peptide. In some embodiments, the first peptide is located downstream of the second peptide.

As used herein, a first peptide being located "upstream" of a second peptide means that the C-terminal residue of the first peptide is located before the N-terminal residue of the second peptide; a first peptide being located "downstream" of a second peptide means that the N-terminal residue of the first peptide is located after the C-terminal residue of the second peptide.

The production and purification of the target polypeptide of the present invention can be achieved under milder pH conditions (e.g., pH 7-11). There are no special restrictions on the type and properties of the target polypeptide. It can be used for the expression and purification of a variety of different polypeptides, and the final output and yield of the target polypeptide are high.

Examples of "target polypeptides" that can be produced and purified by the method of the present invention include, but are not limited to, Bacillus subtilis lipase A (LipA), green fluorescent protein (GFP) and Aspergillus fumigatus type II ketoamine oxidase (AMA), glucagon-like peptide (GLP-1), stromal cell-derived factor (SDF-1α), sermorelin, pleurocidin-like cationic antimicrobial peptide NRC-03 (PNRC03) and Hinnavin II-Melanocyte (HM) or their biologically active fragments.

In one embodiment, the target polypeptide is NsiI protein, for example comprising the amino acid sequence shown in SEQ ID NO: 5.

In one embodiment, the target polypeptide is a TEVP protein, for example comprising the amino acid sequence shown in SEQ ID NO: 8.

In some embodiments, the first peptide and the second peptide in the fusion protein of the present invention are connected by a spacer.

As used herein, "spacer" or "spacer sequence" refers to a peptide of a certain length composed of amino acids with low hydrophobicity and low charge effect, which, when used in a fusion protein, can allow the connected parts to fully unfold and fully fold into their respective natural conformations without interfering with each other. Commonly used spacers in the art include, for example, flexible GS-type linkers rich in glycine (G) and serine (S); and rigid PT-type linkers rich in proline (P) and threonine (T). Since GS-type linkers have a more suitable amino acid length, are hydrophobic and ductile, and can make the functional protein have better stability and biological activity, GS-type linkers are preferably used in the present invention.

In certain applications, such as the production of polypeptide drugs, the polypeptide produced by recombinant production needs to have a consistent sequence with the target polypeptide, i.e., no additional amino acid residues at both ends. For this reason, in some embodiments, the spacer in the fusion protein of the present invention also includes a cleavage site. The cleavage of the cleavage site can release the target polypeptide from the fusion protein.

Suitable cleavage sites include cleavage sites that can be chemically cleaved, enzymatically cleaved or self-cleaved, or any other cleavage sites known to those skilled in the art. Preferred cleavage sites in the present invention can be self-cleaved, for example, they contain the amino acid sequence of a self-cleavable intein. This is because the cleavage method based on intein does not require the addition of enzymes or the use of harmful substances such as hydrogen bromide used in chemical methods, but only requires changing the buffer environment in which the aggregate is located to simply induce cleavage. A variety of self-cleaving inteins are known in the art, such as a series of inteins with different self-cleavage properties from NEB.

In some embodiments, the spacer sequence may contain a specific sequence that can be specifically chemically cleaved or biologically enzymatically hydrolyzed, for example, a Met (methionine) residue can be chemically cleaved by CNBr, a Lys (lysine) or Arg (arginine) residue can be cleaved by trypsin, LysArg or ArgArg can be cleaved by a two-base protease such as Kex2, GluAsnLeuTyrPheGln can be recognized and cleaved by tobacco etch virus protease (TEV protease, TEVP), IleGluGlyArg can be recognized and cleaved by factor Xa (Xa protease), as well as other suitable protease cleavage sites or inteins.

In a further embodiment, the fusion protein may further comprise a portion connected to the second peptide (target polypeptide) for separating and purifying the second peptide, such as a 6-histidine tag, a GST tag, a Strep tag, a Twin-Strep tag, an MBP tag, etc. Thus, after removing the first peptide, the second peptide can be separated and purified by this portion.

In one embodiment, the fusion protein comprises the peptide described in the first aspect of the present invention, a spacer, a polypeptide of interest, and a portion for separating and purifying a second peptide.

In a fourth aspect, the present invention relates to an isolated polynucleotide comprising a nucleotide sequence encoding said fusion protein, and in a fifth aspect, the present invention relates to a construct, in particular an expression construct, comprising the polynucleotide of the fourth aspect.

In the expression construct of the present invention, the sequence of the polynucleotide encoding the fusion protein is operably linked to an expression control sequence to carry out the desired transcription and ultimately produce the fusion protein in a host cell. Suitable expression control sequences include, but are not limited to, promoters, enhancers, ribosome action sites such as ribosome binding sites, polyadenylation sites, transcriptional splicing sequences, transcriptional termination sequences, and sequences that stabilize mRNA, etc.

Vectors used to construct the expression constructs of the present invention include those that replicate autonomously in host cells, such as plasmid vectors; and also include vectors that can be integrated into host cell DNA and replicated with the host cell DNA. Many vectors suitable for the present invention are commercially available. In one embodiment, the plasmid is a plasmid suitable for a prokaryotic or eukaryotic expression system. In a specific embodiment, the plasmid is or is derived from pET28a.

In a sixth aspect, the present invention relates to a host cell capable of expressing the fusion protein of the third aspect of the present invention. The host cell contains a polynucleotide of the fourth aspect of the present invention or a construct of the fifth aspect of the present invention, such as an expression construct, wherein the host cell is capable of expressing the fusion protein.

Host cells for expressing the fusion protein of the present invention include prokaryotes, yeast and higher eukaryotic cells. Exemplary prokaryotic hosts include bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces. In a preferred embodiment, the host cell is an Escherichia cell, a mammalian cell or an insect cell. In a more preferred embodiment, the host cell is Escherichia coli, Bacillus subtilis or Bacillus megaterium. In a specific embodiment of the present invention, the host cell used is an Escherichia coli Rosetta (DE3) strain cell.

A polynucleotide or construct of the invention, such as an expression construct, can be introduced into a host cell to express the encoded amino acid sequence by one of many well-known techniques, including but not limited to heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, liposome-mediated transfection, calcium phosphate precipitation, protoplast fusion, microprojectile bombardment, viral transformation and the like.

In one embodiment, the polynucleotide encoding the fusion protein can be integrated into the genome of a host cell, which can express the encoded fusion protein under appropriate conditions or constitutively express the encoded fusion protein.

In one embodiment, the polynucleotide encoding the fusion protein is present in the host cell in an extrachromosomal form (eg, a plasmid or a construct such as an expression vector).

In a seventh aspect, the present invention also relates to a method for preparing the fusion protein of the third aspect of the present invention, comprising: culturing the host cell of the sixth aspect of the present invention under conditions suitable for expression of the fusion protein; and recovering the fusion protein, optionally, by cutting the fusion protein to release the target polypeptide and recovering the target polypeptide.

In one embodiment, the method comprises: transforming a host cell with a construct, in particular an expression construct, of the fifth aspect of the invention, culturing the transformed host cell under conditions suitable for expression of the fusion protein and recovering the fusion protein, optionally, by cutting the fusion protein to release the target polypeptide and recovering the target polypeptide.

The conditions for expressing fusion proteins are known in the art, such as temperature, pH, culture medium, etc. Any suitable method for recovering fusion proteins is known in the art, including but not limited to, for example, by chromatography, centrifugation, dialysis, etc.

As described herein, the recovery of the fusion protein can be carried out by any suitable method known in the art. For example, after the fusion protein is expressed, the host cells are collected, the cells are lysed, the supernatant is collected (e.g., by centrifugation to remove cell debris), and the fusion protein is optionally separated (e.g., by a specific tag or a specific binding molecule such as an antibody).

In one embodiment, the method comprises: transforming host cells with the construct of the fifth aspect of the invention, in particular the expression construct, culturing the transformed host cells under conditions suitable for expression of the fusion protein, collecting and lysing the cells, collecting the supernatant, and optionally isolating the fusion protein.

In a preferred embodiment, a spacer sequence is included between the short peptide tag of the fusion protein and the target protein, and the spacer sequence contains a cleavage site (such as the cleavage site described herein) that can be cut, thereby removing the short peptide tag by cutting the cleavage site, thereby releasing the target protein.

In an eighth aspect, the present invention provides use of the peptide of the first aspect or the polynucleotide of the second aspect for preparing a target protein in a host cell.

The peptide of the first aspect of the present invention can form a fusion protein with the target protein, so that after being expressed in a host cell, the solubility of the fusion protein can be increased, the expression efficiency of the fusion protein can be improved, and the biological activity of the target protein can be maintained.

For example, the polynucleotide of the second aspect can be linked to a polynucleotide encoding a target protein in frame and placed under the control of a suitable expression regulatory element (eg, a promoter) to express the desired fusion protein in a suitable expression system.

In the ninth aspect, the present invention provides a method for producing a target protein, comprising expressing in a host cell a fusion protein formed by fusion of the peptide described in the first aspect of the present invention and the target protein, such as the fusion protein described in the third aspect of the present invention, recovering the fusion protein, cutting the fusion protein to release the target protein, and optionally isolating and purifying the released target protein.

In one embodiment, the method comprises: constructing an expression construct (e.g., a plasmid or a viral vector) comprising a nucleotide sequence encoding a fusion protein formed by fusion of the peptide described in the first aspect of the present invention and a target protein, transforming a host cell with the expression construct, culturing the transformed host cell under conditions suitable for expression of the fusion protein, recovering the fusion protein, cleaving the fusion protein to release the target protein, and optionally isolating and purifying the released target protein.

In one embodiment, the method comprises: transforming a host cell with the construct of the fifth aspect of the invention, in particular an expression construct, culturing the transformed host cell under conditions suitable for expression of the fusion protein, collecting and lysing the cells, collecting the supernatant, cutting the fusion protein to release the target protein, and optionally isolating and purifying the released target protein.

As described herein, cleavage of the fusion protein can be performed by any suitable means known in the art, such as including a spacer sequence between the short peptide tag and the target protein, wherein the spacer sequence comprises a cleavage site that can be cleaved (such as the cleavage site described herein), thereby removing the short peptide tag by cleaving the fusion protein, thereby releasing the target protein.

In a further embodiment, the fusion protein may further comprise a portion connected to the target protein for separation and purification purposes, such as a 6-histidine tag, a GST tag, a Strep tag, a Twin-Strep tag, an MBP tag, etc. Thus, after removing the first peptide, the target protein can be separated and purified by this portion.

In one embodiment, the method comprises constructing an expression construct comprising a nucleotide sequence encoding the peptide described in the first aspect of the invention, a nucleotide sequence encoding a spacer sequence, a nucleotide sequence encoding a target protein, and a nucleotide sequence encoding a portion for isolating and purifying a second peptide.

In one embodiment, the host cell is selected from prokaryotes, yeasts and eukaryotic cells such as mammalian cells or insect cells, more preferably selected from Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces, more preferably selected from Escherichia, more preferably Escherichia coli, Bacillus subtilis or Bacillus megaterium, more preferably Escherichia coli Rosetta (DE3).

In one embodiment, the target protein has an acidic, neutral or weakly alkaline isoelectric point lower than, equal to or slightly greater than 7.0, for example, equal to or lower than 8.0, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0, and more preferably is an NsiI protein comprising the amino acid sequence shown in SEQ ID NO: 5 or a TEVP protein comprising the amino acid sequence shown in SEQ ID NO: 8.

Compared with the prior art, the present invention has the following beneficial effects:

1) Compared with the traditional technology, the present invention can overcome the disadvantage that the target protein is easy to form inclusion bodies in the prokaryotic expression system, can effectively dissolve the recombinant protein, stabilize the conformation of the recombinant protein during the purification process, improve the expression efficiency of the recombinant protein, maintain its biological function activity, and can be widely used in the fermentation production field of insoluble proteins;

2) The acidic short peptide solubility tag of the present invention can replace the solubility tags such as GST, MBP, and NeonGreen. It is shorter and smaller than the commonly known solubility tags, and has a stable structure. It contains only 57 amino acid residues and a molecular weight of only 8kD. It has less potential impact on the recombinant protein when fused with the recombinant protein;

3) Compared with the previously known positively charged solubilizing tag (Zbasic), the acidic solubilizing tag (Sacid) of the present invention is mutually repelled by the acidic proteins widely present in the body, and is less likely to cause aggregation effects, thereby being more conducive to the dissolution and folding of recombinant proteins, and having more advantages in improving the solubility, expression efficiency and biological activity of recombinant proteins;

4) The tag of the present invention can also obtain high-purity target protein in affinity purification applications, indicating that the modification has no effect on affinity purification.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally included step means that the step exists or does not exist.

As used herein, the term "about" refers to a numerical range that includes a specific value that a person skilled in the art would reasonably consider to be similar to the specific value. In some embodiments, the term "about" refers to within the standard error of measurement using commonly accepted measurements in the art. In some embodiments, approximately refers to +/-10% of a specific value.

Although various embodiments and various aspects of the present invention are shown and described herein, it is apparent to those skilled in the art that these embodiments and various aspects are only examples of the present invention. Those skilled in the art can make various changes, modifications and substitutions without departing from the scope of the spirit of the present invention. It should be understood that the various alternatives of the embodiments of the present invention described herein can be used in the implementation of the present invention.

The present invention will be further described by the following non-limiting examples. It is well known to those skilled in the art that many modifications may be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention.

Unless otherwise specified, the following experimental methods are conventional methods, and the experimental materials used are easily available from commercial companies unless otherwise specified.

The PCR, enzyme digestion, ligation and other experiments involved in conventional plasmid construction, as well as the transformation, bacterial culture and other experiments involved in protein expression are familiar to researchers in this field, so the specific relevant experimental details are not specified in detail. For details, please refer to the conventional experimental conditions described in the Molecular Cloning Experiment Guide [J. Sambrook et al., Molecular Cloning Experiment Guide (3rd Edition) [M], Science Press, 2002].

In addition to the specific methods, equipment, and materials used in the embodiments, based on the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

Example

Example 1: Acid tag design

Based on the amino acid sequence of the B domain of Staphylococcus protein A, as shown in SEQ ID NO: 4, an acidic surface solubilizing peptide tag (Sacid, SEQ ID NO: 1) was designed using computer-aided calculations (URL: https://www.expasy.org/resources/swiss-model), which introduced negatively charged amino acids to concentrate them on the surface. This tag is only a form of predicted acidic tag.

Swiss-Model is a tool for predicting protein structure models. It uses homology modeling to predict the tertiary structure of an unknown sequence.

Example 2: Construction of target protein expression vector

The target protein encoding gene sequence was fused with the acidic surface solubilizing short peptide tag (Sacid) encoding gene sequence to obtain the target protein expression unit (wherein the Sacid amino acid sequence is located upstream of the target protein amino acid sequence), and the target protein expression unit was inserted into the expression plasmid pET-28a (Novagen, USA) to obtain the target protein expression vector pET-28a/Sacid-NsiI by artificial synthesis.

Example 3: Expression of NsiI protein alone

The NsiI nucleotide sequence (encoding amino acid sequence SEQ ID NO: 5) was inserted into the prokaryotic expression vector pET28a to obtain the expression vector pET28a/NsiI. After the vector construction was verified to be correct by sequencing, the plasmid was extracted by alkaline lysis and transformed into the expression host bacteria Rosetta (DE3) (Tiangen Biochemical Technology (Beijing) Co., Ltd.). A single colony of the expression host bacteria containing the recombinant plasmid pET28a/NsiI was inoculated in LB liquid culture containing kanamycin (50μg/mL) and chloramphenicol (34μg/mL) and cultured at 37°C, 220rpm for 16 hours as seed bacteria.

Take the seed bacteria and inoculate them in a sterile LB medium containing kanamycin (50μg/mL) and chloramphenicol (34μg/mL) at a volume ratio of 1:50. Cultivate at 37°C, 200rpm shaking until the appropriate logarithmic growth phase (OD600=0.6-0.8), add 0.5mM IPTG, induce expression at 27°C for 4h-16h, collect the bacterial solution and centrifuge, resuspend the bacterial precipitate in 20mM Tris-HCl, 300mM NaCl pH 8.0 buffer at a concentration of 10%-20%, and ultrasonically disrupt the cells. The lysed bacterial solution was centrifuged at 8000rpm for 30min, the supernatant and precipitate were separated, and SDS-PAGE was performed to identify the expression of the target protein.

The results are shown in Figure 5A. The NsiI target protein was expressed alone, with a molecular weight of about 50 kDa. It was difficult to see the obvious target band at the position indicated by the arrow from SDS-PAGE. Its expression level was not high, the protein was significantly reduced or even completely lost, and there were many impurity proteins after purification.

Example 4: Using NeonGreen as a solubilizing peptide and fusion expression with NsiI

(1) Construction of NeonGreen and NsiI fusion expression vector and host bacteria

The amino acid sequence of NeonGreen used in this experiment is shown in SEQ ID NO: 6.

The genes of NeonGreen and NsiI (SEQ ID NO: 5) and the prokaryotic expression vector pET28a were connected together through artificial synthesis (where the NeonGreen amino acid sequence was located upstream of the NsiI protein amino acid sequence) to obtain the target protein expression vector pET-28a/NeonGreen-NsiI.

(2) Expression and purification of recombinant NeonGreen-NsiI protein

The recombinant plasmid pET-28a/NeonGreen-NsiI was transformed into the expression host bacteria Rosetta (DE3), and a single colony of the expression host bacteria was picked and inoculated into LB liquid culture containing kanamycin (50 μg/mL) and chloramphenicol (34 μg/mL), and cultured at 37°C and 220 rpm for 12 hours as seed bacteria.

Take the seed bacteria and inoculate them in a sterile LB liquid medium containing kanamycin (50μg/mL) and chloramphenicol (34μg/mL) at a volume ratio of 1:50. Cultivate at 37°C and 200rpm with shaking until the appropriate logarithmic growth phase (OD600=0.6-0.8), add IPTG inducer, adjust the temperature to 27°C and culture for 4-16 hours. Centrifuge 200mL of the induced bacterial solution to collect the bacteria, resuspend the bacteria with 30mL 20mM Tris HCl, 300mM NaCl (pH8.0), and then ultrasonically disrupt. Centrifuge the lysed bacterial solution at 8000rpm for 30min, separate the supernatant and precipitate, filter the supernatant with a 0.45uM filter to remove insoluble bacterial proteins, and resuspend the precipitate with deionized water or PBS, and perform SDS-PAGE analysis on the expression product.

(3) Results analysis

As shown in Figure 5B, NeonGreen is used as a solubilizing peptide, and the expression of the NeonGreen-NsiI fusion protein has a molecular weight of about 70KD. The arrow points to the fusion protein. As can be seen from the figure, although the use of NeonGreen as a solubilizing protein fusion expression method can also achieve the purification and enrichment of the fusion protein, due to its large molecular weight, it still has defects such as long folding time, limited solubilizing effect and easy interference with the activity of the recombinant protein. It can be seen from the figure that most of the protein exists in the form of insoluble inclusion bodies and the fusion protein is easily degraded.

Example 5: Fusion expression of published positively charged solubility-enhancing tag (Zbasic) and NsiI

(1) Construction of published positively charged solubilizing tag (Zbasic) and NsiI fusion expression vector and host bacteria

The amino acid sequence of the published positively charged solubilizing tag Zbasic [Hedhammar M, Hober S, Z(basic)--a novel purification tag for efficient protein recovery, Journal of Chromatography A, 2007, 1161(1-2):22-28] is shown as SEQ ID NO: 7. As shown in Example 4, the Zbasic coding sequence and the NsiI coding sequence were connected to obtain a fusion protein expression vector pET-28a/Zbasic-NsiI, wherein the Zbasic amino acid sequence is located upstream of the NsiI amino acid sequence.

(2) Expression and purification of published positively charged solubilizing tags (Zbasic) and NsiI recombinant proteins

The plasmid was transformed into the expression host bacteria Rosetta (DE3) to obtain the target engineered strain. The specific embodiments all adopted laboratory small-scale shake flask expression. Oscillating activation was carried out in LB liquid medium containing kanamycin (50μg/mL) and chloramphenicol (34μg/mL) at 37°C for 16h, and then the overnight culture was transferred to a new LB medium (about 200mL) containing kanamycin (50μg/mL) and chloramphenicol (34μg/mL) at a ratio of 1:50, and cultured at 37°C with shaking until the appropriate logarithmic growth phase (OD600=0.6-0.8), and induced expression at 27°C with 0.5mM IPTG for 4h-16h.

Take 1 mL of the induced bacterial solution, centrifuge at 8000 rpm at 4°C for 2 min, discard the supernatant, collect the bacteria, resuspend them with 200 μL of PBS, and directly add 50 μL of 5×SDS loading buffer (300 mM Tris-HCl (pH 6.8), 20% β-mercaptoethanol, 20% SDS, 25% glycerol, 0.05% bromophenol blue), boil in a boiling water bath for 10 min, and store at -20°C.

Take 200 mL of the induced sample, centrifuge at 8000 rpm at 4°C for 20 min, discard the supernatant, collect the bacteria, resuspend with 30 mL of Tris HCl buffer, add 1% Triton X-100, 1× Cokitail protease inhibitor and 1 mM PMSF, mix well, and ultrasonically disrupt for 30 min. Centrifuge the whole bacterial lysis sample at 12000 rpm at 4°C for 20 min, separate the supernatant and precipitate. Take 40 μL of the supernatant, add 10 μL of 5×SDS loading buffer, boil in water bath for 10 min, and store at -20°C. Resuspend the precipitate with 1 mL of PBS, take 40 μL of it, add 10 μL of 5×SDS loading buffer, boil in water bath for 10 min, and store at -20°C.

The supernatant was purified by affinity chromatography column, 40uL of eluate was added to 10μL of 5×SDS loading buffer, boiled in water bath for 10min, and stored at -20℃. The target protein was purified by Strep Tag II affinity chromatography column, 40uL of eluate was added to 10μL of 5×SDS loading buffer, boiled in water bath for 10min, and stored at -20℃ as the target protein sample. The above samples had the same dilution in electrophoresis detection, and their protein contents were directly comparable.

(3) Protein electrophoresis (SDS-PAGE)

SDS-PAGE gel was prepared according to the conventional method. The prepared protein samples were loaded with equal volumes and tested by 10% SDS-PAGE. The electrophoresis was performed at a constant voltage of 80 V for 30 min, and then at a constant voltage of 125 V. After the electrophoresis, Coomassie Brilliant Blue R-250 staining was performed, and the electrophoresis results were obtained after decolorization.

(4) Results analysis

Figure 5 C shows the NsiI protein expressed using a published positively charged solubilizing tag (Zbasic), with a molecular weight of about 45 kDa, and the arrow indicates the target protein band. As can be seen from Figure 5 D, the protein expression level of the published positively charged solubilizing tag (Zbasic) is relatively poor compared to Sacid.

Example 6: Fusion expression of Sacid and target protein NsiI

(1) Construction of Sacid and NsiI fusion expression vector and host bacteria

The genes of Sacid (SEQ ID NO: 1), NsiI (SEQ ID NO: 5) and the prokaryotic expression vector pET28a were connected together by artificial synthesis to obtain the recombinant expression plasmid pET-28a/Sacid-NsiI expressing the fusion protein, in which the Sacid amino acid sequence was located upstream of the NsiI protein amino acid sequence.

(2) Expression and purification of recombinant Sacid-NsiI

The plasmid was transformed into the expression host bacteria Rosetta (DE3) (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) to obtain the target engineered strain. The specific embodiments all adopted laboratory small-scale shake flask expression. Oscillating activation was carried out in LB liquid culture medium containing kanamycin (50μg/mL) and chloramphenicol (34μg/mL) at 37°C for 16h, and then the overnight culture was transferred to a new LB culture medium (about 200mL) containing kanamycin (50μg/mL) and chloramphenicol (34μg/mL) at a ratio of 1:50, and cultured at 37°C with shaking until the appropriate logarithmic growth phase (OD600=0.6-0.8), and induced expression at 27°C with 0.5mM IPTG for 4h-16h.

(3) Protein electrophoresis (SDS-PAGE)

(4) Results analysis

Figure 5D shows the fusion expression of Sacid-NsiI using the method of the present invention, with a molecular weight of about 45 kDa. There is an obvious target protein band in the purified sample at the position indicated by the arrow. It can be seen from the figure that the expression level of the target protein is greatly improved after the fusion expression of the present invention; compared with other tags, the Sacid tag of the present invention significantly improves the solubility of the fusion protein.

Example 7: Using NeonGreen as a solubilizing peptide and fusion expression with TEVP

(1) Construction of NeonGreen and TEVP fusion expression vector and host bacteria

The genes of NeonGreen and TEVP (SEQ ID NO: 8) and the prokaryotic expression vector pET28a were connected together through artificial synthesis (where the NeonGreen amino acid sequence was located upstream of the TEVP protein amino acid sequence) to obtain the target protein expression vector pET-28a/NeonGreen-TEVP.

(2) Expression and purification of recombinant NeonGreen-TEVP protein

The recombinant plasmid pET-28a/NeonGreen-TEVP was transformed into the expression host bacteria Rosetta (DE3), and a single colony of the expression host bacteria was picked and inoculated into LB liquid culture containing kanamycin (50 μg/mL) and chloramphenicol (34 μg/mL), and cultured at 37°C and 220 rpm for 12 hours as seed bacteria.

Take the seed bacteria and inoculate them in a sterile LB liquid medium containing kanamycin (50μg/mL) and chloramphenicol (34μg/mL) at a volume ratio of 1:50. Cultivate at 37℃ and 200rpm with shaking until the appropriate logarithmic growth phase (OD600＝0.6-0.8), add IPTG inducer, adjust the temperature to 27℃ and culture for 4-16 hours. Centrifuge 200mL of the induced bacterial solution to collect the bacteria, resuspend the bacteria with 30mL 20mM Tris HCl, 300mM NaCl (pH8.0), and then ultrasonically disrupt. Centrifuge the lysed bacterial solution at 8000rpm for 30min, separate the supernatant and precipitate, filter the supernatant with a 0.45uM filter to remove insoluble bacterial proteins, and resuspend the precipitate with deionized water or PBS, and perform SDS-PAGE and Western blot analysis on the expression product.

(3) Result analysis

As shown in Figure 6A, NeonGreen was used as the solubilizing peptide to express the NeonGreen-TEVP fusion protein in the supernatant. The NT molecular weight was about 70KD, and the arrow pointed to the fusion protein. The target bands could not be clearly distinguished from the SDS PAGE in Figure A. In order to better observe the two bands, the proteins were subjected to Western blotting. The results are shown in Figure 6B, NT. From the results of Western blotting, it can be seen that the NeonGreen-TEVP fusion protein was not expressed.

Example 8: Fusion expression of Sacid and target protein TEVP

(1) Construction of Sacid and TEVP fusion expression vector and host bacteria

The genes of Sacid (SEQ ID NO: 1), TEVP (SEQ ID NO: 8) and the prokaryotic expression vector pET28a were connected together through artificial synthesis (where the Sacid amino acid sequence was located upstream of the TEVP protein amino acid sequence) to obtain the recombinant expression plasmid pET-28a/Sacid-TEVP.

(2) Expression and purification of recombinant Sacid-TEVP

(3) Protein electrophoresis (SDS-PAGE) and Western blotting

The proteins on the gel were wet transferred to a PVDF membrane, blocked with 5% skim milk powder for 2 h, incubated with primary antibody at 4°C overnight, incubated with HRP-labeled secondary antibody at room temperature for 1 h, and exposed using a chemiluminescence kit and GelView 6000Plus equipment. The target band is shown in ST in Figure 6B.

(4) Results analysis

Lane ST in Figures 6A and B is Sacid-TEVP fusion expressed by the method of the present invention, with a molecular weight of about 39 kDa. The arrow indicates the position where there is an obvious target protein band in the supernatant sample and the Western blotting results are consistent with it.

It can be seen from FIG6 that the expression level of the target protein is greatly improved after the fusion expression of the present invention; compared with other tags, the Sacid tag of the present invention significantly improves the solubility of the fusion protein.

Example 9: Purified protein activity test

The obtained protein eluate was subjected to ultrafiltration to replace the buffer, and the protein was stored in Tris HCl buffer (20mM Tris HCl, 20mM NaCl, 50% glycerol), and the protein activity was verified by enzyme digestion of the plasmid.

The plasmid containing a single NsiI restriction site and a single HindIII restriction site was used as a substrate for double restriction digestion. The purified NsiI enzyme was diluted in a gradient (10 ¹ to 10 ⁸ times) and then subjected to double restriction digestion reaction. Each dilution was used as a sample. The enzymes were digested for 1 hour at 37°C and then subjected to agarose gel electrophoresis. The enzyme and plasmid dosages were the same and comparable.

Table 1 Double enzyme digestion reaction system

The results are shown in Figure 7. Using a plasmid containing a single NsiI restriction site and a single HindIII restriction site as a substrate, after double enzyme digestion reaction, target bands of 3392bp and 1987bp were obtained. Figure A shows the double enzyme digestion identification of the NsiI protein expressed alone and the NeonGreen-NsiI protein; Figure B shows the double enzyme digestion identification of the NsiI protein expressed by the published positive charge solubility tag (Zbasic) and the Sacid-NsiI protein of the present invention. It can be seen from the figure that the NsiI protein expressed alone is almost inactive; the activities of the NsiI protein expressed by the NeonGreen-NsiI fusion protein and the published positive charge solubility tag (Zbasic) are basically the same; and the activity of the purified protein Sacid-NsiI is greatly improved by using the technology of the present invention with Sacid as the purification tag, which is higher than the activity of the NsiI protein expressed by the NeonGreen-NsiI fusion protein and the published positive charge solubility tag (Zbasic).

By measuring the specific activity of the enzyme, the results are shown in Figure 8. The technology of the present invention uses Sacid as a purification tag, and the enzyme activity of the purified protein Sacid-NsiI is 496KU/mg, the enzyme activity of the NsiI protein expressed by the known positively charged solubilizing tag (Zbasic) is 5.5KU/mg, and the enzyme activity of the NeonGreen-NsiI fusion protein is 4.9KU/mg. The NsiI protein expressed alone is almost inactive and has the problem of contamination by foreign proteins. As can be seen from the figure, the technology of the present invention uses Sacid as a purification tag, and the enzyme activity of the purified protein Sacid-NsiI is about 100 times that of the enzymes purified by the other two known solubilizing tags.

SEQ ID NO：1(Sacid)

Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Asp-Ala-Phe-Tyr-Glu-Ile-Leu-His-Leu-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Phe-Ile-Gln-Glu-Leu-Lys-Asp-Asp-Pro-Glu-Glu-Ser-Asp-Glu-Glu-Leu-Glu-Glu-Ala-Asp-Asp-Leu-Asn-Asp-Ala-Gln-Pro-Lys

SEQ ID NO：2(Sacid 1)

Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Asp-Ala-Phe-Tyr-Glu-Ile-Leu-His-Leu-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Phe-Ile-Arg-Ser-Leu-Arg-Asp-Asp-Pro-Ser-Gln-Ser-Ala-Asn-Leu-Leu-Ala-Glu-Ala-Asp-Asp-Leu-Asn-Asp-Ala-Gln-Pro-Lys

SEQ ID NO: 3 (Sacid 2)

Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Arg-Arg-Arg-Ala-Phe-Tyr-Glu-Ile-Leu-His-Leu-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Phe-Ile-Arg-Ser-Leu-Arg-Asp-Asp-Pro-Ser-Gln-Ser-Ala-Asn-Leu-Leu-Ala-Glu-Ala-Asp-Asp-Leu-Asn-Asp-Ala-Gln-Pro-Lys

SEQ ID NO: 4 (B domain of Staphylococcus protein A, Wt)

Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Gln-Gln-Asn-Ala-Phe-Tyr-Glu-Ile-Leu-His-Leu-Pro-Asn-Leu-Asn-Glu-Glu-Gln-Arg-Asn-Ala-Phe-Ile-Gln-Ser-Leu-Lys-Asp-Asp-Pro-Ser-Gln-Ser-Ala-Asn-Leu-Leu-Ala-Glu-Ala-Lys-Lys-Leu-Asn-Asp-Ala-Gln-Pro-Lys

SEQ ID NO: 5(NsiI)

Met-Ile-Asn-His-Ser-Ile-Leu-Lys-His-His-Ser-Phe-Thr-Gly-Lys-Ile-Ile-Ser-Ile-Leu-Lys-Asp-Glu-Phe-Gly-Asp-Asp-Ala-Ile-Tyr-Ile-Phe-Glu-Asn-Ser-Pro-Ile-Leu-Gly-Tyr-Leu-Asn-Ile-Lys-Thr-Lys-Ser-Ala-Glu-Arg-Gly-Ser-Lys-Ser-Arg-Gly-Ser-Phe-Ala-Asn-His-Tyr-Ala-Leu-Tyr-Val-Ile-Ile-Glu-Asp-Tyr-Ile-Asn-Lys-Gly-Tyr-Leu-Gly-As p-Asp-Leu-Asp-Tyr-Ser-Lys-Tyr-Asp-Gly-Ala-Lys-Phe-Thr-Asp-Leu-Phe-Arg-Arg-Gln-Arg-Glu-Leu-Pro-Phe-Gly-Ser-Lys-Leu-Gln-Asn-His-Ala-Leu-Asn-Ser-Arg-Leu-Asn-Asp-Glu-Phe-Lys-Lys-Phe-Phe-Pro-Thr-Leu-Gly-Ile-Val-Pro-Ile-Ile-Arg-Asp-Val-Arg-Thr-Ser-Arg-Tyr-Trp-Ile-Gln-Glu-Asp-Leu-Ile-Lys-Val-Ser-Val-Arg-Asn-Ly s-Asn-Gly-Ile-Glu-Arg-Arg-Glu-Asn-Leu-Ala-Pro-Ser-Ile-Ile-Arg-Ile-Ile-Asp-Glu-Tyr-Ile-Ala-Thr-Lys-Lys-Glu-Ser-Phe-Glu-Leu-Phe-Leu-Lys-Thr-Cys-Gln-Glu-Ile-Ala-Asn-Leu-Ser-Ser-Ser-Asp-Pro-His-Ser-Val-Val-Lys-Phe-Ile-Gln-Glu-Gln-Leu-His-Pro-Ser-Ser-Asp-Ala-Arg-Val-Phe-Glu-Ile-Val-Ser-Tyr-Ala-Val-Leu-Lys-Gl u-Arg-Tyr-Ser-Asn-Gln-Thr-Ile-Trp-Ile-Gly-Asp-Ser-Arg-Asp-Asp-Val-Ala-Glu-Glu-Ser-Leu-Val-Leu-Tyr-Lys-Thr-Gly-Arg-Thr-Asn-Ala-Asn-Asp-Gly-Gly-Ile-Asp-Phe-Val-Met-Lys-Pro-Leu-Gly-Arg-Phe-Phe-Gln-Val-Thr-Glu-Thr-Ile-Asp-Ala-Asn-Lys-Tyr-Phe-Leu-Asp-Ile-Asp-Lys-Val-Gln-Arg-Phe-Pro-Ile-Thr-Phe-Val-Val-Lys-Th r-Asn-Ser-Ser-Tyr-Glu-Glu-Ile-Glu-lys-Ile-Ile-Lys-Glu-Gln-Ala-Lys-Ala-Lys-Tyr-Asn-Ile-Glu-Ala-Ile-Val-Asn-Ser-Tyr-Met-Asp-Ser-Ile-Glu-Glu-Ile-Ile-Asn-Val-Pro-Asp-Leu-Met-Lys-Tyr-Phe-Glu-Glu-Met-Ile-Tyr-Ser-Asp-Ser-Leu-Lys-Arg-Ile-Met-Asp-Glu-Ile-Ile-Val-Gln-Ser-Lys-Val-Glu-Phe-Asn-Tyr-Glu-Glu-Asp-Val-Ser

SEQ ID NO: 6 (NeonGreen)

Met-Leu-Ser-Lys-Gly-Glu-Glu-Asp-Asn-Met-Ala-Ser-Leu-Pro-Ala-Thr-His-Glu-Leu-His-Ile-Phe-Gly-Ser-Ile-Asn-Gly-Val-Asp-Phe-Asp-Met-Val-Gly-Gln-Gly-Thr-Gly-Asn-Pro-Asn-Asp-Gly-Tyr-Glu-Glu-Leu-Asn-Leu-Lys-Ser-Thr-Lys-Gly-Asp-Leu-G ln-Phe-Ser-Pro-Trp-Ile-Leu-Val-Pro-His-Ile-Gly-Tyr-Gly-Phe-His-Gln-Tyr-Leu-Pro-Tyr-Pro-Asp-Gly-Met-Ser-Pro-Phe-Gln-Ala-Ala-Met-Val-Asp-Gly-Ser-Gly-Tyr-Gln-Val-His-Arg-Thr-Met-Gln-Phe-Glu-Asp-Gly-Ala-Ser-Leu-Thr-Val-Asn-Tyr-Arg -Tyr-Thr-Tyr-Glu-Gly-Ser-His-Ile-Lys-Gly-Glu-Ala-Gln-Val-Lys-Gly-Thr-Gly-Phe-Pro-Ala-Asp-Gly-Pro-Val-Met-Thr-Asn-Ser-Leu-Thr-Ala-Ala-Asp-Trp-Cys-Arg-Ser-Lys-Lys-Thr-Tyr-Pro-Asn-Asp-Lys-Thr-Ile-Ile-Ser-Thr-Phe-Lys-Trp-Ser-Tyr-T hr-Thr-Gly-Asn-Gly-Lys-Arg-Tyr-Arg-Ser-Thr-Ala-Arg-Thr-Thr-Tyr-Thr-Phe-Ala-Lys-Pro-Met-Ala-Ala-Asn-Tyr-Leu-Lys-Asn-Gln-Pro-Met-Tyr-Val-Phe-Arg-Lys-Thr-Glu-Leu-Lys-His-Ser-Lys-Thr-Glu-Leu-Asn-Phe-Lys-Glu-Trp-Gln-Lys-Ala-Phe-Thr

SEQ ID NO: 7 (Zbasic)

Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Arg-Arg-Arg-Ala-Arg-Arg-Glu-Ile-Arg-His-Leu-Pro-Asn-Leu-Asn-Arg-Glu-Gln-Arg-Arg-Ala-Phe-Ile-Arg-Ser-Leu-Arg-Asp-Asp-Pro-Ser-Gln-Ser-Ala-Asn-Leu-Leu-Ala-Glu-Ala-Lys-Lys-Leu-Asn-Asp-Ala-Gln-Pro-Lys

SEQ ID NO: 8 (TEVP)

Lys-Gly-Pro-Arg-Asp-Tyr-Asn-Pro-Ile-Ser-Ser-Ser-Ile-Cys-His-Leu-Thr-Asn-Glu-Ser-Asp-Gly-His-Thr-Thr-Ser-Leu-Tyr-Gly-Ile-Gly-Phe-Gly-Pro-Phe-Ile-Ile-Thr-Asn-L ys-His-Leu-Phe-Arg-Arg-Asn-Asn-Gly-Thr-Leu-Val-Val-Gln-Ser-Leu-His-Gly-Val-Phe-Lys-Val-Lys-Asp-Thr-Thr-Thr-Leu-Gln-Gln-His-Leu-Val-Asp-Gly-Arg-Asp-Met-Ile-Ile-Ile-Arg-Met-Pro-Lys-Asp-Phe-Pro-Pr o-Phe-Pro-Gln-Lys-Leu-Lys-Phe-Arg-Glu-Pro-Gln-Arg-Glu-Glu-Arg-Ile-Cys-Leu-Val-Thr-Thr-Asn-Phe-Gln-Thr-Lys-Ser-Met-Ser-Ser-Met-Val-Ser-Asp-Thr-Ser-Cys-Thr-Phe-Pro-Ser-Gly-Asp-Gly-Ile-Phe-Trp-Lys- His-Trp-Ile-Gln-Thr-Lys-Asp-Gly-Gln-Cys-Gly-Ser-Pro-Leu-Val-Ser-Thr-Arg-Asp-Gly-Phe-Ile-Val-Gly-Ile-His-Ser-Ala-Ser-Asn-Phe-Thr-Asn-Thr-Asn-Asn-Tyr-Phe-Thr-Ser-Val-Pro-Lys-Asn-Phe-Met-Glu-Leu-Le u-Thr-Asn-Gln-Glu-Ala-Gln-Gln-Trp-Val-Ser-Gly-Trp-Arg-Leu-Asn-Ala-Asp-Ser-Val-Leu-Trp-Gly-Gly-His-Lys-Val-Phe-Met-Val-Lys-Pro-Glu-Glu-Pro-Phe-Gln-Pro-Val-Lys-Glu-Ala-Thr-Gln-Leu-Met-Asn-Glu-Gly.

Claims

An isolated peptide comprises an amino acid sequence VDNKFNKEQQX 1 AFYEILHLPNLNEEQRNAFIQX 2 LKDDPX 3 X 4 SX 5 X 6 X 7 LX 8 EAX 9 X 10 LNDAQPK (SEQ ID NO: 9), wherein: X 1 -X 10 are all negatively charged amino acids, preferably E or D,

Preferably, the isoelectric point pi of the peptide is equal to or less than 5.0.
The peptide of claim 1, which comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of the amino acid sequence shown in SEQ ID NO: 1.
An isolated polynucleotide encoding the peptide of claim 1 or 2.
An isolated fusion protein comprises a first peptide and a second peptide, wherein the first peptide is the peptide of claim 1 or 2, the second peptide is a target polypeptide, and optionally the second peptide is linked to the first peptide via a spacer.
The fusion protein of claim 4, wherein:

The spacer comprises a cleavage site, preferably selected from a chemical cleavage site, an enzymatic cleavage site and a self-cleavage site, and/or,

The fusion protein further comprises a part connected to the second peptide for separation and purification, such as a 6-histidine tag, a GST tag, a Strep tag, a Twin-Strep tag or an MBP tag, and/or,

The second peptide has an acidic, neutral or weakly basic isoelectric point lower than, equal to or slightly greater than 7.0, for example, equal to or lower than 8.0, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0. More preferably, the length of the second peptide is 20-500 amino acid residues, for example, an NsiI protein comprising the amino acid sequence shown in SEQ ID NO: 5 or a TEVP protein comprising the amino acid sequence shown in SEQ ID NO: 8.
An isolated polynucleotide comprising a nucleotide sequence encoding the fusion protein of claim 4 or 5.
A construct, preferably an expression construct, comprising the polynucleotide of claim 6.
A host cell comprising the polynucleotide of claim 6 or the construct of claim 7, preferably an expression construct, wherein the host cell is capable of expressing the fusion protein, preferably, the host cell is selected from prokaryotes, yeasts and eukaryotic cells such as mammalian cells or insect cells, more preferably selected from Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces, more preferably selected from Escherichia, more preferably Escherichia coli, Bacillus subtilis or Bacillus megaterium, more preferably Escherichia coli Rosetta (DE3).
A method for producing the fusion protein of claim 4 or 5, comprising:

(a) culturing the host cell of claim 8 under conditions suitable for expression of the fusion protein; and

(b) recovering the fusion protein,

Optionally,

(c) cleaving the fusion protein to release the polypeptide of interest; and

(d) recovering the target polypeptide,

Preferably, the target polypeptide has an acidic, neutral or weakly basic isoelectric point lower than, equal to or slightly greater than 7.0, for example equal to or lower than 8.0, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0, and more preferably is an NsiI protein comprising the amino acid sequence shown in SEQ ID NO: 5 or a TEVP protein comprising the amino acid sequence shown in SEQ ID NO: 8.
A method for producing a target polypeptide, comprising:

(a) expressing a fusion protein formed by fusion of a target polypeptide and the peptide of claim 1 or 2 in a host cell, wherein the peptide of claim 1 or 2 is preferably located upstream of the target polypeptide;

(b) recovering and cleaving the fusion protein to release the polypeptide of interest; and

(c) optionally, isolating and purifying the target polypeptide,

Preferably, the target polypeptide has an acidic, neutral or weakly basic isoelectric point lower than, equal to or slightly greater than 7.0, for example equal to or lower than 8.0, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0. More preferably, the target polypeptide has a length of 20-500 amino acid residues, more preferably a NsiI protein comprising the amino acid sequence shown in SEQ ID NO: 5 or a TEVP protein comprising the amino acid sequence shown in SEQ ID NO: 8.
The method of claim 10, wherein:

The polypeptide of interest is linked to the peptide of claim 1 or 2 via a spacer, preferably the spacer comprises a cleavage site, preferably selected from a chemical cleavage site, an enzymatic cleavage site and a self-cleavage site, and/or

The fusion protein further comprises a part connected to the target polypeptide for separation and purification, such as a 6-histidine tag, a GST tag, a Strep tag, a Twin-Strep tag or an MBP tag.
The method of any one of claims 9 to 11, wherein the host cell is selected from prokaryotes, yeasts and eukaryotic cells such as mammalian cells or insect cells, more preferably selected from Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces, more preferably selected from Escherichia, more preferably Escherichia coli, Bacillus subtilis or Bacillus megaterium, more preferably Escherichia coli Rosetta (DE3).
Use of the peptide of claim 1 or 2 or the polynucleotide of claim 3 for preparing a target protein in a host cell, wherein the host cell is preferably selected from prokaryotes, yeasts and eukaryotic cells such as mammalian cells or insect cells, more preferably selected from Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces, more preferably selected from Escherichia, more preferably Escherichia coli, Bacillus subtilis or Bacillus megaterium, more preferably Escherichia coli Rosetta (DE3),

Preferably, the target protein has an acidic, neutral or weakly alkaline isoelectric point lower than, equal to or slightly greater than 7.0, for example equal to or lower than 8.0, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0. More preferably, the target protein has a length of 20-500 amino acid residues, more preferably a NsiI protein comprising the amino acid sequence shown in SEQ ID NO: 5 or a TEVP protein comprising the amino acid sequence shown in SEQ ID NO: 8.