CN111019926B - TEV protease variants, fusion proteins thereof, and methods of making and using - Google Patents

Abstract

The invention provides TEV protease variants, fusion proteins thereof, and methods of making and using. The invention provides TEV protease variants and fusion proteins with unique properties which have been screened for. The TEV protease variant and the polypeptide fusion expression can be used for quickly and efficiently preparing the polypeptide and solving the problems in the current recombinant polypeptide production process.

Description

TEV protease variants, fusion proteins thereof, and methods of making and using

The present application claims the benefit of priority of chinese patent application No.201811177281.2 filed on 10 months and 10 days in 2018.

Technical Field

The invention relates to the field of proteins, in particular to TEV protease.

Background

The TEV Protease (TEV Protease) is an active structural domain of 27kDa in Nla Protease derived from Tobacco Etch Virus (TEV), and the amino acid sequence of the TEV Protease is shown in SEQ ID NO. 1.TEV protease has strong site specificity and can recognize EXXYXQ (G/S) heptaamino acid sequence, the most commonly used sequence is Glu-Asn-Leu-Tyr-Phe-Gln-Gly (or ENLYFQG), the cleavage site is between glutamine Gln (P1) and glycine Gly (P1 ') (i.e., between P1 and P1'), and the sequence specificity is far higher than that of proteases such as thrombin, factor Xa, enterokinase, etc.

TEV protease is able to tolerate a wide range of pH (pH 4-8.5) and temperature (4-34 ℃) and to varying degrees some common additives that increase protein solubility or stability (glycols, EGTA, detergents and reducing agents). It has been demonstrated that TEV protease is insensitive to ethylene glycol, EGTA and some detergents (Triton X-100, Tween-20 and NP-40), decreases in activity in the presence of 1% CHAPS, and still retains most of the activity in low concentrations of denaturants (2M urea, 1% SDS) and reductants (0.7M. beta. -mercaptoethanol) (C.Sun et al, 2012).

Since wild-type TEV enzyme has some defects in expression, solubility, and the like, a number of mutants improved by genetic engineering techniques have been reported. For example, the native TEV protease undergoes self-cleavage, which is continuously subjected to conformational changes due to collisions with other TEV proteases during expression and purification, and self-cleavage occurs at a specific site, so that the intact protease is truncated and its activity is greatly reduced. The S219N mutant found by Lucast et al has a greatly improved stability, but is not highly soluble, with about 95% of the protein being present in the pellet as inclusion bodies. Kapust et al point-mutated the gene sequence of the native TEV protease using genetic engineering to obtain a mutant of S219V, which has high stability and slightly improved enzyme activity, and which has about 100 times higher stability than S219N. Second, TEV protease expression yields are not high, and solubility is also very low. Approximately only 5% of TEV protease was present in the supernatant of the cell disruption solution, yielding 12.5 mg/L. Van den Berg et al found a mutant TEVSH that could increase the yield to 54mg/L by gene shuffling (DNA shuffling) and error-prone PCR (error-prone PCR), and had much improved solubility over S219N and little change in enzyme activity. Cabrita, L.D. et al use PoPMuSiC software design to perform stability analysis on TEV protease single-point mutants, and screen out five mutants, the solubility and enzyme activity of which are both improved compared with wild type TEV protease, and a double mutant is also obtained, and the solubility and enzyme activity of which are significantly improved compared with the single mutant. In response to the shortcomings of TEV protease itself, researchers have been looking for improved mutants. For example, the TEV Ser135Gly mutant is more stable than WT and can tolerate exposure to higher temperatures (>40 ℃); still other mutations (T17S, N68D, N177V) may significantly improve the solubility of TEV protease.

TEV protease has found widespread use as a tool enzyme in protein research and biopharmaceutical production. The reaction conditions may be selected according to the nature of the protein of interest. By designing a recombinant TEV protease with its own histidine tag, the TEV protease can also be easily cleared after cleavage using the affinity tag. For example, TEV protease is often used to cleave fusion proteins with high efficiency, but is limited to cleavage in a state where both are soluble. The mainstream scheme for preparing protein polypeptide drugs by cutting fusion protein by TEV protease at present is as follows: the polypeptide and a tag protein (such as MBP) are fused and expressed, and are connected by enzyme digestion sequences; purifying the fusion protein; preparing TEV protease; cleaving the fusion protein with TEV protease; and (4) separating and purifying the enzyme digestion product. The production method has the defects of excessive process links, low efficiency and unfavorable cost control.

In response to the above-described problems in the recombinant production process of polypeptides, the inventors have found a TEV protease variant which is suitable for the rapid and efficient production of polypeptides according to the present invention.

Disclosure of Invention

The inventor finds that the process can be greatly simplified by adopting a mode of directly fusing and expressing TEV protease and polypeptide to prepare the polypeptide medicament. At present, the conventional TEV protease can cut a large amount of fusion protein (self-digestion) in the expression process, so that the polypeptide is released in advance, is easy to be hydrolyzed by intracellular protease, is not beneficial to purification, and is not suitable for industrial production. Although most of the activity of conventional TEV protease can still be maintained in low concentration denaturants (2Murea, 1% SDS) (c.sun et al, 2012), many proteins are often not completely soluble under low concentration denaturation conditions such as 2M urea, which limits the use of TEV protease. In addition, although the small-scale laboratory preparation of TEV protease is low in cost, the large-scale preparation of TEV protease requires large-scale fermentation, purification and renaturation, and the production cost is still high.

The invention provides TEV protease variants and fusion proteins with unique properties which have been screened for. The fusion protein can be used for quickly and efficiently preparing polypeptide, and solves the problems in the prior recombinant polypeptide production process.

The present invention is based, in part, on the fact that the TEV protease variants of the present invention have no or very weak protease cleavage activity in cells during expression, but have very good enzymatic cleavage activity in vitro under conditions of high degree of denaturation.

In one aspect, the invention provides TEV protease variants.

In one embodiment, the TEV protease variant may have low enzymatic activity during expression in a host. Preferably, the TEV protease variant has a lower enzymatic activity during expression in a host compared to the S219V variant having the amino acid sequence shown in SEQ ID No. 10.

In one embodiment, the TEV protease variant may have high protease activity under conditions of high or moderate degree of denaturation. Preferably, the moderate to high degree of denaturing conditions are in an in vitro environment of 3M to 5M urea, preferably 3.5M to 4.5M urea, more preferably 4M urea or 1M to 2M guanidine hydrochloride, preferably 1.5M guanidine hydrochloride. Preferably, the TEV protease variant retains the in vitro enzymatic activity of the S219V variant under conditions of high degree of denaturation.

In one embodiment, enzymatic activity is determined for a fusion protein comprising a TEV protease variant and its cleavage site. Preferably, the cleavage site is selected from the group consisting of EXXYXQG/S/H, wherein X is any amino acid residue, preferably the cleavage site is selected from the group consisting of SEQ ID NO 7 and 8. In one embodiment, the fusion protein may comprise the structure TEVp-sTEV-Y1, wherein Y1 is a polypeptide of interest; TEVp is a TEV protease variant; sTEV is a TEV protease cleavage site, which is EXXYXQG/S/H, wherein X is any amino acid residue, preferably said cleavage site is selected from the group consisting of SEQ ID NO 7 and 8.

In one embodiment, the TEV protease variant may comprise one or more mutations selected from the group consisting of:

a mutation from leucine (L) to phenylalanine (F) or histidine (H) at a position corresponding to position 111 of the sequence represented by SEQ ID NO. 1;

a mutation of isoleucine (I) to lysine (K) at a position corresponding to position 138 of the sequence represented by SEQ ID NO. 1;

a mutation from histidine (H) to leucine (L) at a position corresponding to position 28 of the sequence represented by SEQ ID NO. 1;

a mutation from glutamic acid (Q) to histidine (H) at a position corresponding to position 196 of the sequence represented by SEQ ID NO. 1;

a mutation from serine (S) to glycine (G) at a position corresponding to position 135 of the sequence represented by SEQ ID NO. 1; and

a mutation of methionine (M) to isoleucine (I) at a position corresponding to position 187 of the sequence represented by SEQ ID NO: 1.

In one embodiment, the TEV protease variant may comprise a combination of mutations selected from:

a mutation from leucine (L) to phenylalanine (F) at a position corresponding to position 111 of the sequence represented by SEQ ID NO.1 and a mutation from isoleucine (I) to lysine (K) at a position corresponding to position 138 of the sequence represented by SEQ ID NO. 1;

a mutation from histidine (H) to leucine (L) at a position corresponding to position 28 of the sequence represented by SEQ ID NO.1, a mutation from leucine (L) to phenylalanine (F) at a position corresponding to position 111 of the sequence represented by SEQ ID NO.1, and a mutation from glutamic acid (Q) to histidine (H) at a position corresponding to position 196 of the sequence represented by SEQ ID NO. 1; and

a mutation from leucine (L) to histidine (H) at a position corresponding to position 111 of the sequence represented by SEQ ID NO:1, a mutation from serine (S) to glycine (G) at a position corresponding to position 135 of the sequence represented by SEQ ID NO:1, and a mutation from methionine (M) to isoleucine (I) at a position corresponding to position 187 of the sequence represented by SEQ ID NO: 1.

In one embodiment, the TEV protease variant may comprise a mutation from serine (S) to valine (V) at a position corresponding to position 219 of the sequence shown in SEQ ID NO: 1.

In one embodiment, the TEV protease variant may further comprise one or more mutations other than the above-described mutations, provided that said TEV protease variant has a low enzymatic activity during in-host expression, preferably a lower enzymatic activity during in-host expression compared to the S219V variant having the amino acid sequence shown in SEQ id No.10 and/or said TEV protease variant has a high enzymatic activity under conditions of medium to high degree of denaturation, preferably in an in vitro environment of 3M to 5M urea, preferably 3.5M to 4.5M urea, more preferably 4M urea or 1M to 2M guanidine hydrochloride, preferably 1.5M guanidine hydrochloride, preferably the TEV protease variant retains the in vitro enzymatic activity of the S219V variant under conditions of medium to high degree of denaturation.

In one embodiment, the protease variant may comprise the amino acid sequence shown in SEQ ID NO 4, 5 or 6 or homologues thereof.

In one embodiment, the homologue may comprise an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID No. 4, 5 or 6. Homologues should retain the above-mentioned properties of the TEV protease variants.

In one embodiment, the homologue may comprise an amino acid sequence having a substitution, deletion or addition of at least 1, preferably at least 2, more preferably at least 3, most preferably at least 4 amino acid positions to SEQ ID No. 4, 5 or 6. Homologues should retain the above-mentioned properties of the TEV protease variants.

In one embodiment, the homologue may have low enzymatic activity during expression in the host, preferably the homologue has lower enzymatic activity during expression in the host compared to the S219V variant having the amino acid sequence shown in SEQ ID No.10 and/or the TEV protease variant has high enzymatic activity under conditions of medium to high degree of denaturation, preferably in an in vitro environment of 3M-5M urea, preferably 3.5M-4.5M urea, more preferably 4M urea or 1M-2M guanidine hydrochloride, preferably 1.5M guanidine hydrochloride, preferably the homologue retains the in vitro enzymatic activity of the S219V variant under conditions of medium to high degree of denaturation.

In one embodiment, the homologue is derived from tobacco etch virus.

In one aspect, the invention provides fusion proteins, which may comprise the TEV protease variants of the invention. Unexpectedly, the inventors have found that incorporation of the TEV protease variants of the invention into the fusion proteins of the invention allows for the rapid and efficient production of polypeptides of interest.

In one embodiment, the fusion protein may comprise the structure TEVp-sTEV-Y1,

wherein Y1 is a polypeptide of interest;

TEVp is according to the aforementioned TEV protease variant;

sTEV is a TEV protease cleavage site, which is EXXYXQG/S, wherein X is any amino acid residue, preferably said cleavage site is selected from the group consisting of SEQ ID NO 7 and 8.

In one embodiment, the polypeptide of interest can be selected from ACTH, GLP-1/GLP-2, IFN- α, IFN- γ, Histatin, CCL5, SDF-1 α, IGF-1, Leptin, BNP, Ex-4, preferably ACTH, preferably human ACTH, more preferably human ACTH having the amino acid sequence set forth in SEQ ID No. 2.

In one embodiment, the TEVp and sTEV may be directly linked or separated by one or more amino acid residues, provided that the TEVp recognizes and cleaves the sTEV.

In one embodiment, sTEV and Y1 may be directly linked or separated by one or more amino acid residues, provided that TEVp is capable of recognizing and cleaving sTEV.

In one embodiment, the fusion protein may further comprise a tag.

In one embodiment, the tag may be a purification tag.

In one embodiment, the tag may be selected from the group consisting of: a His tag, a Maltose Binding Protein (MBP) tag, a glutathione transferase (GST) tag, a NusA tag, a SUMO tag, an Avi tag, a T7 tag, an S tag, a Flag tag, an HA tag, a c-myc tag, or a strepII tag.

In one embodiment, the tag may be at the N-terminus of the fusion protein.

In one embodiment, the N-terminal of the TEVp is unlabeled.

In one aspect, the invention provides a polynucleotide sequence encoding a TEV protease variant of the invention or a fusion protein of the invention. The polynucleotide sequence is preferably selected from SEQ ID NO. 14-16.

In one aspect, the invention provides a polynucleotide construct comprising a polynucleotide sequence of the invention.

In one aspect, the invention provides an expression vector comprising a polynucleotide sequence of the invention or a polynucleotide construct of the invention.

In one embodiment, the expression vector may be a eukaryotic expression vector or a prokaryotic expression vector.

In one embodiment, the expression vector may be a eukaryotic expression vector. Preferably, the eukaryotic expression vector may be selected from the group consisting of: pRS314, pYES2, baculovirus-S2 expression system and pcDNA3.1.

In one embodiment, the expression vector may be a prokaryotic expression vector. Preferably, the prokaryotic expression vector may be selected from the group consisting of: pET series expression vector, pQE series expression vector and pBAD series expression vector.

In one aspect, the invention provides a cell comprising a polynucleotide sequence of the invention, a polynucleotide construct of the invention, or an expression vector of the invention.

In one embodiment, the cell may be a eukaryotic cell or a prokaryotic cell.

In one embodiment, the cell may be a eukaryotic cell. Preferably, the eukaryotic cell may be selected from the group consisting of: saccharomyces cerevisiae, insect cell expression system.

In one embodiment, the cell may be a prokaryotic cell. Preferably, the prokaryotic cell may be selected from the group consisting of: BL21, BL21(DE3), BL21(DE3) pLysS, Rosetta2, Rosetta2pLysS, Tuner (DE3), or Origami 2.

In one aspect, the present invention provides a method of making a TEV protease variant of the present invention, comprising:

(1) culturing the cells of the invention in a culture medium under conditions suitable for culturing the cells;

(2) harvesting the culture medium, or lysing the cells to harvest the lysate;

(3) purifying to obtain the TEV protease variant.

In one aspect, the present invention provides the use of a TEV protease variant for the preparation of a polypeptide of interest, wherein said TEV protease variant is expressed as a fusion protein with said polypeptide of interest. Preferably, the fusion protein is a fusion protein of the invention.

In one aspect, the invention provides a method of making a polypeptide of interest, comprising:

(1) culturing the fusion protein of the present invention in a medium under suitable conditions;

(2) obtaining inclusion bodies of the fusion protein;

(3) solubilization of the inclusion bodies under highly denaturing conditions, e.g., about 8M urea or about 6M guanidine hydrochloride;

(4) incubating under conditions of medium to high degree of denaturation, preferably under 3M to 5M urea, preferably 3.5M to 4.5M urea, more preferably 4M urea or 1M to 2M guanidine hydrochloride, preferably 1.5M guanidine hydrochloride, at a temperature, for example 20 to 40 ℃, preferably 25 ℃ for a period of time, for example 10 to 24 hours, for example 12 hours;

(5) precipitating the TEV protease after dilution with a buffer such as Tris-HCl, preferably 50mM Tris-HCl;

(6) removing the TEV protease precipitate to obtain the polypeptide of interest; and is

(7) Purifying the polypeptide of interest.

In one embodiment, the purification may be performed using a technique selected from the group consisting of: salting out, ultrafiltration, organic solvent precipitation, gel filtration, ion exchange chromatography column, and reversed phase high performance liquid chromatography.

The invention has the advantages that:

1. the TEV protease variants of the invention can be widely used to produce a variety of polypeptides or proteins having a native N-terminus.

2. The TEV protease variants of the invention are obtained by screening, but directly obtained mutants do not meet the requirements or partially meet the requirements. Improved mutants with a unique further improved performance of the gain can be obtained by a series of combinations of mutation points, for example L111F in combination with other sites, characterized by no or very low activity in vivo but higher activity under moderately denaturing conditions in vitro.

3. The method adopts a DNA recombination technology and a prokaryotic expression system to express the target protein, and has wide applicability.

4. Compared with the method for extracting the ACTH from the pig pituitary, the production cost of the human-derived or pig-derived ACTH prepared by the method disclosed by the invention is reduced by hundreds of times compared with the traditional pig brain extraction production technology, the production period is greatly shortened, the production time and cost are saved, the pig pituitary is not required to be obtained by slaughtering a large number of live pigs, the risks of immunogenic anaphylactic reaction and the like caused by the use of the pig-derived ACTH are avoided, and the safety of the medicine is greatly improved. The ACTH prepared by the method is completely the same as ACTH secreted by a human body, has extremely high amino acid sequence precision and product purity, and excellent biological activity, improves the curative effect of the medicine, and reduces the occurrence of adverse reactions. Compared with a chemical organic synthesis method, the method has the advantages that the difficulty and the cost of preparing the full-length human ACTH are obviously reduced, the operation is simple, expensive catalysts and high-pressure equipment are not needed, the yield is high, and the method is suitable for large-scale production. In general, the method has the advantages of clear and simple production process, good repeatability, easy realization of large-scale production and reduction of environmental pollution.

Drawings

FIG. 1: SDS-PAGE gel Coomassie blue staining of TEVP mutant 12D for cleavage efficiency in different concentrations of urea. Arrows indicate the target protein.

FIG. 2: SDS-PAGE gel Coomassie blue staining of TEVP mutant 4D for cleavage efficiency in urea at different concentrations. Arrows indicate the target protein.

FIG. 3: SDS-PAGE gel Coomassie blue staining of TEVP mutant 32C for cleavage efficiency in urea at different concentrations. Arrows indicate the target protein.

FIG. 4A: SDS-PAGE gel Coomassie blue staining of TEVP mutant 4D for cleavage efficiency in urea at different concentrations. FIG. 4B: and comparing the gray values of the electrophoretic bands.

FIG. 5: the in vivo cleavage activity and in vitro cleavage activity of TEVP mutants 4D, 12D and 32C were compared with those of S219V. Arrows indicate the target protein.

FIG. 6: solubility of the fusion proteins of TEVP mutants 4D, 12D, 32C and S219V in 0.5M urea was examined.

FIG. 7: plasmid pET-28b-PD1-Avi map.

FIG. 8: plasmid pET-28b-ACTH map.

FIG. 9: plasmid Avi-TEV-sTEV-ACTH Gene map.

FIG. 10: the mass spectrometer measures the molecular weight of the purified ACTH.

FIG. 11: ACTH activity was measured by in vitro method.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below, but the embodiments of the present invention are not limited thereto.

TEV protease

TEV protease, although often used to cleave fusion proteins efficiently, is limited to cleavage in a state where both are soluble. Most of the current polypeptide drugs are poorly soluble and are usually expressed in the form of inclusion bodies, which are not compatible with the cleavage conditions of TEV protease. Inclusion bodies need to be dissolved by high concentration urea or guanidine hydrochloride, but enzyme digestion under such conditions is very challenging, and conventional TEV protease and substrate are difficult to have both high-efficiency cleavage activity and simultaneous dissolution under the same conditions.

The inclusion body is beneficial to purification, and relatively pure target protein can be obtained by simple treatment. However, the inclusion body protein needs to be dissolved by high-concentration urea or guanidine hydrochloride, and the wild-type TEV protease has no activity or extremely low activity under the condition, so that the fusion protein cannot be efficiently cut. At present, a great deal of improved TEV protease is reported, but the optimal enzyme cutting conditions are physiological conditions or non-denaturing conditions, and the improved TEV protease generally has certain tolerance to a denaturant (urea or guanidine hydrochloride) with low concentration, but most of the inclusion body protein is insoluble under the denaturing condition with low concentration, so that the conventional TEV protease is not suitable for the occasion.

The inventors found that the ideal production conditions for preparing polypeptide medicaments by fusing TEV protease and polypeptide are as follows: (1) the fusion protein is expressed in the form of inclusion body, which is beneficial to purification and can protect the protein from being hydrolyzed by protease; (2) the fusion protein does not undergo self-cleavage in cells, otherwise, the purification is not facilitated; (3) after being dissolved in a high-concentration denaturant, the fusion protein is diluted into an enzyme digestion reaction system containing the high-concentration denaturant, and at the moment, the TEV protease recovers the activity, self-cleaves efficiently and releases polypeptide; (4) the TEV protease variant obtained by screening and evolving from a high-capacity TEV protease library has a broad spectrum of amino acids at the P1' position of the enzyme cutting site, so that the TEV protease variant can be widely used for producing various polypeptides or proteins with natural N ends.

Method of the invention

The structure of a fusion protein with self-cleavage, which is usually prepared, is provided in US2010035300a 1. In the preparation of fusion proteins, a His tag is required to be closely linked to the protein of interest (e.g., EGFP) to be expressed. The reason for this is that when the fusion protein is expressed in vivo, the conventional wild-type TEVp or mutant TEVp has a certain enzymatic activity in the cell, and self-cleavage occurs. Cleaved EGFP can be recovered via His tag that is closely linked thereto. Without this His tag, it is difficult to recover the EGFP by self-cleavage in the cell, and thus EGFP cannot be efficiently produced. However, this also poses a problem in that the His tag of EGFP may be further removed as required. This is obviously time consuming and laborious.

In the present invention, the target protein in the fusion protein may not contain a tag, such as His. In one embodiment, the fusion protein comprises the structure of Avi-TEVp-sTEV-ACTH, where Avi is the Avi tag protein and sTEV is the TEV protease cleavage site. Because TEVp variants (e.g., 12D variants) have very low intracellular enzymatic activity, the fusion protein that can be expressed is not substantially cleaved intracellularly. When the inclusion bodies were collected, solubilized with 8M urea, and conditioned to an environment of 4M urea, 12D exhibited its enzymatic activity, and the recognition site was cut to sTEV. After the enzyme digestion is finished, the final concentration of urea or guanidine hydrochloride is reduced to 0.5M through simple dilution, TEV protease is easy to precipitate due to poor solubility, and the released polypeptide with biological activity is easy to dissolve in a buffer solution, so that the high-purity polypeptide can be obtained through centrifugation, the original protease removal link needing to be added is ingeniously overcome, and the later-stage purification process is greatly simplified.

The formation of inclusion bodies is more favorable for purification: 1) high-concentration and relatively pure protein can be easily harvested by centrifugation; 2) inclusion bodies protect proteins from proteolytic hydrolysis. In addition, the toxic protein is expressed in an inactive inclusion body form, and the growth of host bacteria is not influenced. For example, the TEVP-ACTH fusion protein involved in the method is expressed in the form of inclusion bodies, the purification steps are greatly simplified, higher concentration and purity can be achieved, and the protein is not hydrolyzed by protease, so that stable and high yield can be easily obtained.

ACTH production method of the present invention

The method of the invention adopts a mode of directly fusing and expressing the TEV protease and the ACTH polypeptide to prepare the polypeptide drug, and the method depends on a unique TEV protease variant and can greatly simplify the process. The conventional TEV protease can cut a fusion protein in a large amount during the expression process (self-digestion), for example, the MBP-TEVP fusion protein is used for preparing TEVP, which can lead to the premature release of the polypeptide, and the purification is difficult, so that the method is not suitable for industrial production. The invention provides a method for preparing protein polypeptide medicaments based on fusion proteins of unique TEV protease variants and application thereof. This TEV protease variant has the following characteristics: (1) the enzyme digestion activity in vivo is very low, about 0-10%, the polypeptide fused and expressed with TEV is guaranteed not to be released in advance, and meanwhile, the polypeptide yield can be increased; (2) the fusion protein is expressed in the form of inclusion body, greatly simplifying the purification steps, achieving higher concentration and purity, and being not hydrolyzed by protease, thus being easy to obtain stable high yield; (3) after the inclusion body is dissolved in 8M urea or 6M guanidine hydrochloride, the inclusion body is directly diluted into a 4M urea or 1.5M guanidine hydrochloride enzyme digestion buffer solution, and at the moment, the TEV protease recovers the activity, self-cleaves efficiently and accurately, and ACTH polypeptide is released; (4) after the enzyme digestion is finished, the final concentration of urea or guanidine hydrochloride is reduced to 0.5M through simple dilution, and the soluble ACTH polypeptide, the insoluble TEV protease and the fusion protein which is not digested can be separated through centrifugation. Compared with the conventional preparation of polypeptide drugs by fusion proteins, the whole process is greatly simplified, the separation of enzyme digestion products from the protein surface is achieved, and a primary product with relatively high purity can be obtained by only four steps.

The purpose of the invention is realized by the following technical scheme: a fusion protein of a TEV protease variant and an ACTH polypeptide, having the structural formula: Avi-TEVP-sTEV-ACTH. Wherein, the Avi is Avi label protein, a short peptide of 15 amino acids, and a single biotinylation lysine site; TEVP is a TEV protease variant; sTEV is a TEV protease restriction site, and the sequence of the sTEV is as follows: l-glutamic acid 1-L-aspartic acid 2-L-leucine 3-L-tyrosine 4-L-phenylalanine 5-L-glutamine 6-L-serine; ACTH is a human ACTH polypeptide, the amino acid sequence of which is shown in SEQ ID No. 2. The invention connects protease enzyme digestion amino acid sequence (sTEV) between TEV protease and target polypeptide, integrates the fusion protein expression and protease expression into the same step, and expresses in the form of inclusion body, thus not only can express a large amount, but also can obtain fusion protein with relatively high purity by simple treatment (ultrasonic crushing, washing, centrifuging) (under physiological condition in cell, TEVP variant has no enzyme digestion activity or extremely weak activity due to poor solubility, and can not self-cut in large amount), and after dissolving inclusion body with 8M urea, the fusion protein is directly diluted to medium-high concentration denaturation condition (4M urea, wild type TEV protease has lower activity under the condition), and then enzymatic hydrolysis process can be started to release polypeptide. After the enzymolysis product is diluted, TEV protease is easy to precipitate due to poor solubility, and the released polypeptide with biological activity is easy to dissolve in buffer solution, so that high-purity polypeptide can be obtained by centrifugation, the original protease removing link needing to be added is ingeniously overcome, and the later-stage purification process is greatly simplified. The TEV protease variant can be used as a platform for preparing protein polypeptide medicaments by a fusion protein method, is used for quickly and efficiently expressing and purifying polypeptides which are difficult to realize by a conventional recombinant expression method and a chemical synthesis method or have high cost, and is particularly suitable for preparing polypeptides which are easy to degrade or have toxicity to host bacteria. The technical platform can greatly reduce the time and cost of the downstream process, and is suitable for large-scale industrial production and small-scale laboratory preparation.

Determination of the cleavage Activity or efficiency

The in vivo enzyme digestion activity can be calculated according to the result of SDS-PAGE gel, and the specific process is as follows: diluting an inclusion body of the TEV protease variant by 10 times, adding a 5xSDS loading buffer solution containing 10% beta-mercaptoethanol, boiling the sample at 100 ℃ for 5min, loading and running the gel, putting the gel into a Coomassie brilliant blue staining solution to stain for 30min, then putting the gel into the Coomassie brilliant blue destaining solution, and heating and destaining for about 20min until the background is colorless. The gel was then placed in a gel imaging system for photography. The obtained pictures were processed with Image J software, and the band grayscale values were quantified, with the in vivo cleavage activity being 1-fusion protein band grayscale value/(fusion protein band grayscale value + Avi-TEVP band grayscale value fusion protein molecular weight/Avi-TEVP molecular weight).

Herein, the method of analyzing the gray values of the electrophoretic bands using Image J software is currently a general method.

In this context, the in vitro cleavage efficiency is calculated as follows:

in vitro enzyme cutting efficiency is 1-band gray value of the fusion protein after enzyme cutting/band gray value of the fusion protein before enzyme cutting.

Herein, urea 8M or more or guanidine hydrochloride 6M or more is considered as a high concentration of denaturant, while urea 4M is a medium concentration of denaturant. The medium-high degree of denaturation conditions are 4M urea or 1.5M guanidine hydrochloride, 50mM Tris-HCl (pH8.0), 1mM EDTA,2mM DTT, reaction temperature 4-37 deg.C, preferably 25 deg.C. Preferably 3M to 5M urea, preferably 3.5M to 4.5M urea, more preferably 4M urea or 1M to 2M guanidine hydrochloride, preferably 1.5M guanidine hydrochloride, at a temperature, e.g.20 to 40 ℃, preferably 25 ℃ for a period of time, e.g.10 to 24 hours, e.g.12 hours.

The following examples are provided to illustrate the invention.

Examples

Example 1: obtaining TEV protease variants

1. Large volume TEV protease random mutation library construction

1.1 Experimental materials:

coli TG 1: supE hsd Δ 5thi Δ (lac-proAB) F' [ traD36proAB + lacIqlacZ Δ M15], purchased from Weian, Kyoto, Baokada, Beijing. Phagemid vector pHEN1 (purchased from Biovector NTCC plasmid vector bacterial cell Gene Collection, Cat. Biovector 786623). DNA polymerase, T4DNA ligase and restriction enzyme were purchased from EnxWeiji trading Ltd. The plasmid extraction kit and the agarose gel DNA recovery kit are purchased from Tiangen (Beijing) Biotechnology Ltd. Random Mutagenesis Kit (GeneMorph II Random Mutagenesis Kit) was purchased from Agilent Technologies. Primer synthesis and gene sequencing were performed in Nanjing Kingsrei Biotech, Inc.

TEVP random mutation library construction

1.2.1 random mutagenesis PCR preparation of TEVP DNA fragments

Using pQE30-TEV (S219V) (this 27kDa TEV NIa protease variant gene was synthesized by Nanjing Kirsi, cloned between BamH1 and HindIII of pQE30 vector, S219V amino acid sequence shown in SEQ ID NO.10, nucleotide sequence shown in SEQ ID NO.3) as a template, TEVP S219V gene was subjected to extensive Random Mutagenesis using Random Mutagenesis Kit (GeneMorph II Random Mutagenesis Kit). The PCR primers are:

TEV-F：5-

AATCTCGAGGGATCTAAAGGTCCTGGAGAAAGCTTGTTTAAGGGACCAC-3

TEV-R：5-AATGGATCCTTGCGAGTACACCAATTC-3

the specific steps are operated according to the instruction, the PCR cycle number and the template dosage are well controlled, and the PCR condition is set as the condition for generating the medium mutation. The 50ul reaction system was prepared as follows: to the PCR tube were added, 41.5. mu.l ddH2O, 5. mu.l 10 XMutazyme II reaction buffer, 1. mu.l 40mM dNTP mix (final concentration 200. mu.M each), 0.5. mu.l primer mix (250 ng/. mu.l each), 1. mu.l Mutazyme II DNA polymerase (2.5U/. mu.l), 1. mu.l TEVP template (template amount 10ng), PCR amplification conditions: pre-denaturation at 95 ℃ for 3 min; at 95 ℃ for 30 s; 30s at 60 ℃; 72 ℃, 50s, 32 cycles, and finally 72 ℃ extension for 10 min. After PCR is finished, 5ul of product is taken out and is run on 1% agarose gel for electrophoresis detection, the 1.1-kb gel standard substance in the kit for molecular weight standard is used, after dyeing, the brightness of a target band and the standard substance is compared, the PCR yield is calculated, and the amplification multiple is obtained by dividing the PCR yield by the template amount, so that the d value in the PCR process is calculated (calculation formula: 2)^dYield of PCR/initialTemplate amount). The final d value of the TEVP random mutation PCR of the present invention is 7.5, and the corresponding mutation frequency is about 9 mutations/kb (refer to the specification), which meets the expected requirements. The PCR product was purified by phenol chloroform extraction, double digested with Xho1 and BamH1, and the digested product was recovered with agarose gel DNA recovery kit and stored at-20 ℃.

1.2.2 preparation of linearized vector

The linearized phagemid vector pHEN1 (purchased from Biovector NTCC plasmid vector bacterial cell Gene Collection, Catalogue number Biovector786623) was modified before it was used. First, an Avi tag sequence GLNDIFEAQKIEWHE was inserted downstream of the signal peptide, and the fusion protein thus produced was biotinylated and then immobilized on streptavidin magnetic beads. PCR amplification of Avi tagged template Using pET28b-PD1-Avi (synthesized in Kinsry, Nanjing, with the gene structure Nco1-PD1 gene-Not 1-BamHI-GGGS linker-Avi tag-Xho 1, Genebank accession No. NM-005018 for PD1 gene, plasmid map see FIG. 7), the PCR primers were as follows:

Avi-F:5-ACTCCATGGCCGGTCTGAATGATATTTTTGAAGC-3

Avi-R:5-AATCTCGAGCTCGTGCCACTCGATTTTCTG-3

the product was purified with phenol chloroform, double digested with Nco1 and Xho1, and the gel recovered and ligated with the same digested pHEN1 vector to give pHEN 1-Avi. Then, a tandem sequence comprising sTEV (nucleotide sequence: GAAAATCTGTATTTTCAGAGC, amino acid sequence ENLYFQS) and human ACTH gene was inserted behind Avi tag (cloning site Xho1+ Not1), the template for PCR amplification of the sTEV-ACTH tandem sequence was pET28b-ACTH (synthesized in Kingkunshire, plasmid map shown in FIG. 8, amino acid sequence shown in SEQ ID NO. 2; nucleotide sequence shown in SEQ ID NO.13), and PCR primers were as follows:

ACTH-F:

AATCTCGAGGGATCTGGATCCGGAGGTGGCGGTAGCGAAAATCTGTATTTTCAGAGCTATAGCATGGAAC-3

ACTH-R:5-AATGCGGCCGCAAATTCCAGCGGAAATGC-3

additional BamH1 site was introduced upstream of the primer, and the PCR product was purified with phenol chloroform, double digested with Not1 and Xho1, and ligated with the same digested pHEN1-Avi vector to give pHEN 1-Avi-sTEV-ACTH. And (3) vector linearization: the pHEN1-Avi-sTEV-ACTH was double digested with Xho1 and BamH1, the large fragment of the digested product was recovered by gel, and stored at-20 ℃.

1.2.3 ligation and Recycling of ligation products

Connecting the vector (pHEN1-Avi-sTEV-ACTH) recovered by enzyme digestion and the insert (randomly mutated TEVP gene) according to the ratio of 1:3, 1:5 and 1:10, and carrying out the specific process: the vector pHEN1-Avi-sTEV-ACTH was prepared (see preparation of 1.2.2 linearized vector), and the insert was prepared (see preparation of TEVP DNA fragment by 1.2.1 random mutagenesis PCR), and the two were ligated by T4DNA ligase (Thermo Fisher Scientific) to obtain pHEN 1-Avi-TEVp-sTEV-ACTH. The connection system is referred to the specification, taking 20ul reaction system as an example: the PCR tubes were filled sequentially with 150ng of linearized vector, the corresponding amount of insert (molar ratio 1:3 or 1:5 or 1:10 to vector), 10 XT 4DNA Ligase buffer 2ul, T4DNA Ligase 1ul and finally filled up to 20ul with ddH 2O. The optimal ligation ratio was determined to be 1:5 by counting the number of clones produced by transformation. The plasmid map is shown in FIG. 9. The optimal ligation ratio was determined to be 1: 5. The transformation was performed with different concentrations of ligation product to determine the optimal amount of transformation product to be 0.2. mu.g (10. mu.l). The ligation product was purified by phenol chloroform extraction to remove protein and salt ions and dissolved in ddH 2O.

1.2.4 electrotransformation

The ligation product was transferred to the host strain TG1 by electroporation. Electrotransformation competent cells were prepared according to the molecular cloning protocol (third edition). And (3) taking 10 mu l of the ligation product, gently mixing with 200 mu l of the electroporation competent cells, carrying out ice bath for 2min, transferring into an electroporation cuvette with a precooled pore space of 0.2cm, and carrying out electroporation. The parameters of the electrotransfer instrument (Bio-Rad Gene-Pulser) were 2.5KV, 25. mu.F, 200. omega. Immediately adding 1ml of SOC culture medium after electric shock, rinsing the electric shock cup for 2 times by using 1ml of SOC culture medium after sucking out, combining 3ml of bacterial liquid, and shaking for 45-60 min at 37 ℃ and 200 rpm. Diluting the bacterial liquid in a gradient manner: 10^-2，10^-3,10^-4100ul of the cells were applied to a small SOC (containing 25. mu.g/ml carbenicillin) plate per gradient, the remaining cells were collected by centrifugation at 5000Xg for 5min, and the SOC (containing 25. mu.g/ml carbenicillin) plate was applied after resuspension) A square large plate (25x25cm), carrying out inverted culture at 37 ℃ for 12-16 h, counting by using a small plate, washing bacterial colonies on the large plate by using a 2YT culture medium, slightly and uniformly scraping by using a coating rod, adding glycerol to a final concentration of 50%, subpackaging into small tubes, and freezing and storing at-80 ℃. Library capacity calculation method: number of clones on the plates dilution multiple total volume of pre-diluted broth. Repeating the electrotransfer for multiple times until the library capacity reaches 1x10⁹The above.

1.2.5 sequence determination and analysis

Randomly selecting a plurality of original bacterial clones, verifying recombinant plasmids by colony PCR, and carrying out PCR verification primers:

upstream primer 5-CCACCATGGCCGGTCTGAATGATATTTTTGAAGC-3

A downstream primer: 5-TTGTTCTGCGGCCGCAAATTCCAGC-3

The PCR-positive recombinant plasmids were sent for sequencing (Kinry Biotechnology, Inc.) and the constructed libraries were evaluated for randomness, capacity and abundance.

1.2.6 library capacity and diversity evaluation

The molar ratio of the vector to the insert was 1:5, 10. mu.l of the ligation product was electrotransferred with a transformation efficiency of 9X10⁸cfu/. mu.g DNA. After combining multiple transformations, the library volume was 2.02x10⁹And meets the requirement of screening. 20 clones were randomly picked from the peptide library and sequenced. The results show (Table 1) that the overall mutation frequency was low or medium (1.5-7/kb). The actual value has a certain deviation from the theoretical value, probably because the synonymous mutation is not counted, and because the sample is smaller, the difference is gradually reduced along with the increase of the sequencing sample. The results show that the constructed TEVP random phage library meets the design requirements from the basic framework, the library capacity and the diversity.

TABLE 1 distribution of randomly selected cloned amino acid mutations

2. Screening of target clones from TEVP random mutant phage libraries

2.1 materials of the experiment

HRP-M13 antibody was purchased from Beijing Yiqiao Shenzhou, 96-well ELISA plate and ELISA reagent were purchased from Shanghai. Streptavidin magnetic beads were purchased from NEB. Reagents such as D-biotin, IPTG and the like are purchased from Shanghai.

2.2 phage library culture

At least one library volume of bacteria was added to a 2L Erlenmeyer flask containing 2xYT-CG (2xYT + 100. mu.g/ml carbenicillin and 2% glucose) to an initial OD600 of about 0.1, shaken at 37 ℃ and 200rpm until the OD600 reached about 0.5, infected (MOI of 20) with the helper phage M130K07 (from Ann. Co., Beijing Baokoushi)) and incubated at 37 ℃ for one hour in 100 rpm slow rotation. Subsequently, the cells were harvested by centrifugation (2000Xg for 20 minutes), the supernatant was removed and discarded, and the cells were resuspended in 1L of 2XYT-CK (2XYT + 100. mu.g/ml carbenicillin and 50. mu.g/ml kanamycin + 200. mu.M D-biotin) and incubated at 25 ℃ overnight at 250 rpm. The next day, the packaged phage were harvested and purified. The cell culture was transferred to a 750 ml centrifuge bottle and cooled slightly in ice, centrifuged at 4500 rpm for 30 minutes at 4 ℃ using a large bench top centrifuge, if the supernatant was cloudy, it was transferred to another clean bottle and the procedure repeated. The supernatant 80% (without agitation of the cell pellet) was transferred to another new 750 ml centrifuge bottle, 1/6 volumes of PEG/NaCl solution (20% [ w/v ] PEG-8000, 2.5M NaCl) was added, mixed, then allowed to stand on ice for at least two hours, centrifuged at 4500 rpm for 30 minutes at 4 deg.C, carefully removed all supernatant, 10 ml PBS was added, the pellet was redissolved with a pipette and then transferred to a 2ml Ep tube. The cells were centrifuged at 15000Xg for 20 minutes at 4 ℃ to remove the remaining cells or debris. Carefully pipette the supernatant into a fresh Ep tube, add 1/6 volumes of PEG/NaCl solution, stand on ice for 1 hour to re-precipitate phage, then centrifuge at 15000Xg for 20 minutes at 4 ℃ to collect the phage pellet, carefully pipette off and discard all supernatants. Subsequently, the phage pellet was redissolved with an appropriate amount of PBS, and after complete solubilization, centrifuged at 15000Xg for 10 minutes at 4 ℃ to remove remaining insoluble impurities. Subpackaging the supernatant into new EP tubes, adding 50% glycerol, and storing at-80 deg.C for a long period.

2.3 determination of library Titers

Reference Carol M Y Lee et al (M Y Lee, Carol)&Iorno,Niccoló&Sierro,Frederic&Christ, Daniel. (2007). Selection of human antibodies fragments by phase display. Nature protocols.2.3001-8.10.1038/nprot.2007.448.) phage libraries were diluted with a 2XYT gradient, 10 were taken^-9，10^-10，10^-11Adding 10ul of each dilution into 200ul of fresh TG1 bacterial solution cultured to logarithmic phase, mixing, incubating at 37 ℃ for 30min, and coating 2 XYT-CG-containing plates. The mixture was incubated overnight at 30 ℃ in an incubator, and the single colony count was counted the next day to calculate the titer.

2.4 library panning

Principle of panning for TEVP variants of the invention: avi tag is a short peptide tag (GLNDIFEAQKIEWHE) consisting of 15 amino acid residues, and biotin is linked to a lysine residue by biotin ligase in vivo or in vitro, so that biotinylation of protein is realized, and the biotinylated protein can be specifically bound by streptavidin. Under ideal conditions, the TEVP variant with enzyme digestion activity in cells can be subjected to enzyme digestion in the expression process, so that the terminal of the finally assembled phage PIII protein only has ACTH, has no Avi tag, and cannot be captured by magnetic beads; and the TEVP variant without enzyme cleavage activity does not carry out self-digestion in the expression process when the protein is expressed in cells, the N end of the finally assembled phage PIII protein is Avi-TEVP-sTEV-ACTH, and the Avi tag at the N end is biotinylated and can be captured by magnetic beads containing streptavidin. And (3) incubating the phage captured by the magnetic beads in high-concentration urea, wherein TEVP variants which are not dissolved under physiological conditions can be dissolved and recovered in activity under the conditions, and performing enzyme digestion, wherein the phage subjected to enzyme digestion under the conditions can fall off from the magnetic beads due to self-cleavage and enter a solution. The solution is collected to obtain the initial target phage, and the amplified phage may be screened in the next round. After several rounds of screening, TEV protease variants with weak in vivo enzymatic activity and enzymatic activity under in vitro denaturing conditions are enriched. The enriched sequence is obtained by gene sequencing analysis and cloned to an expression vector for verification one by one. The panning for each round is shown in table 2 below.

TABLE 2 panning data for each round

The specific process of phage panning is as follows:

(1) phage fixation: in a clean 2ml EP tube, 100 times the library capacity of phage diluted into TBST buffer (50mM Tris-HCl pH7.5, 150mM NaCl, 0.1% [ v/v ] Tween-20), adding appropriate streptavidin magnetic beads mixed, 4 degrees C rotation incubation for 20min, using the magnet to precipitate magnetic beads, then TBST washing magnetic beads 5 times, remove the unbound phage.

(2) Enzyme digestion screening: adding TBS buffer solution containing 3M urea into the magnetic beads, incubating at room temperature for 1-2h, precipitating the magnetic beads by using a magnet, and collecting all supernatant as much as possible.

(3) Phage amplification: the eluted phage was added to 50ml of TG1 host bacteria with OD600 ═ 0.5, slowly shaken at 37 ℃ for 1 hour, added with 100 μ g/ml of carbenicillin and 2% glucose, cultured for 2 hours, added with helper phage (M130K07 (from "ann" of beijing bacco)) to infect (MOI ═ 20), and cultured at low rpm for 1 hour at 37 ℃. Subsequently, the cells were harvested by centrifugation, the supernatant was removed and discarded, and the cells were resuspended in 100ml of 2XYT-CK (2XYT + 100. mu.g/ml carbenicillin and 50. mu.g/ml kanamycin + 200. mu.M D-biotin) and incubated at 25 ℃ overnight at 250 rpm. The next day, the packaged phage were harvested and purified. The culture was transferred to a clean 50ml centrifuge tube and centrifuged at 13,000g for 20min at 4 ℃. The upper 80% of the supernatant was transferred to a fresh centrifuge tube, 1/6 volumes of PEG/NaCl solution was added and the mixture was allowed to settle at 4 ℃ for more than 1 hour. Then centrifuging again to collect the precipitate, adding a proper amount of PBS to resuspend the phage precipitate, centrifuging again to remove impurities such as bacterial debris, collecting the supernatant, adding 1/6 volumes of PEG/NaCl solution, standing on ice for 1 hour, then centrifuging again to collect the precipitate, dissolving the precipitate in a proper amount of PBS, centrifuging for 10 minutes at 13,000g to remove insoluble impurities, and transferring the supernatant to another fresh Ep tube, namely the eluate after amplification.

(4) Mu.l of purified phage was removed for titer determination and the rest was used for the next round of panning or storage.

(5) Sequencing analysis screening for sequence enrichment: after 3 rounds of panning, the phage eluted in the last round was spread on a 2XYT-CG plate after infecting host bacteria, and cultured overnight at 30 ℃. The next day, the single colony is picked, colony PCR is firstly carried out to determine whether the colony belongs to the library sequence, and the positive clone is sent to sequencing analysis. Through analyzing the sequencing result, the occurrence frequency of the mutation sites is counted, and finally, the best 7 mutants are selected to be used as candidate clones.

3. Candidate clone characterization

The 7 candidate sequences were cloned into the expression vector pET28b (from Novagen) by digestion (Nco1/Not1) and transformed into Rosetta2(DE3) (from Youbao, Hunan) for expression. The expression conditions are as follows: the cells were cultured in LB medium at 37 ℃ until OD600 became about 0.6. Induction conditions are as follows: after shaking at 250rpm at 37 ℃ and 2mM IPTG, the induction was carried out for 4 hours. And (3) bacterium treatment: centrifuging to collect bacteria, carrying out resuspension, crushing by using ultrasonic waves, centrifuging to collect an inclusion body precipitate, washing the inclusion body, and finally dissolving the inclusion body by using 8M urea (50mM Tris-HCl,1mM EDTA,2mM DTT,8M urea, pH8.0).

And (3) diluting the inclusion body protein and performing enzyme digestion test: the inclusion body protein of each candidate clone was diluted with TEVP digestion buffer (50mM Tris-HCl,1mM EDTA,2mM DTT, pH8.0) to a final urea concentration of 3M or 4M, and then digested overnight at 25 ℃. The next day, the restriction was checked by running SDS-PAGE gel. The protein electrophoretic bands were processed with Image J software and the band grey values were calculated.

Optimizing enzyme cutting conditions: the positive clones obtained by the above digestion test were further tested for digestion efficiency under various conditions including: different urea concentrations, different temperatures, different guanidine hydrochloride concentrations, DTT and EDTA concentrations in the reaction solution.

Through the screening, 3 mutants with weak enzyme digestion activity in vivo but normal activity in vitro are finally obtained. The test results are shown in FIGS. 1, 2, 3, 4A-B.

The specific process comprises the following steps: each of the TEV protease variant-ACTH fusion proteins dissolved in 8M urea buffer was added at a ratio of 1:10 to a dilution of 50mM Tris-HCl pH8.0, 1mM EDTA,2mM DTT containing urea at various concentrations. For a final Urea concentration of 4M, the dilution was 3.56M Urea, 50mM Tris-HCl pH8.0, 1mM EDTA,2mM DTT. Each sample was mixed well and immediately aliquoted into two EP tubes, one of which was immediately added with 5 XSDS-loading buffer and cooked at 100 ℃ for 5min, which was a sample digested for 0 h. The other sample was subjected to self-digestion at 25 ℃ for 16h, then 5 XSDS-loading buffer was added, and the sample was boiled at 100 ℃ for 5min, which was a sample digested for 16 h. Subsequently, the enzyme-cleaved 0h, 16h samples of each variant were examined by running SDS-PAGE denaturing gel. After electrophoresis, the gel was stained with Coomassie brilliant blue, destained and photographed. The pictures were processed with Image J software and the electrophoretic band gray values were calculated. The calculation method is as follows:

in vivo cleavage activity ═ 1-fusion protein band gray value before cleavage/(fusion protein band gray value before cleavage + Avi-TEVP band gray value x fusion protein molecular weight/Avi-TEVP molecular weight).

In vitro enzyme cutting efficiency is 1-band gray value of the fusion protein after enzyme cutting/band gray value of the fusion protein before enzyme cutting.

As shown in Table 3, the 4D, 12D and 32C protease variants showed a significant reduction in vivo cleavage activity, with cleavage efficiencies above 30% in 4M urea in vitro. The TEV protease variants used in the preparation of ACTH polypeptides of the invention are any of table 3. The TEV protease variants screened by the present invention can be used for the preparation of other recombinant polypeptides and proteins.

Table 3: the yields of the fusion proteins of several protease variants and ACTH (calculation method: running a fusion protein sample and a BSA standard product together on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) denatured gel, after running the gel, placing the gel into Coomassie brilliant blue staining solution for staining, then transferring the stained solution into a destaining solution for background removal, taking a picture, comparing the gray value of the BSA standard product band in the picture with the gray value of the sample to estimate the concentration of the sample, further calculating the concentration and yield of the fusion protein), the in vivo activity and the in vitro tolerance capacity to 4M urea (using S219V as a control), and comparing based on the gray value of the protein band. Data are statistics of the yields and in vivo and in vitro enzyme-cleavage ratios of fusion proteins from 3 different batches of TEVP variants expressed.

Note: the yield of the fusion protein is the fusion protein which is not subjected to enzyme digestion, and the enzyme digested part in vivo is not calculated because of no utilization value. S219V is a mutation from serine (S) to valine (V) at amino acid 219 of wild type TEVP;

4D: the 111 th amino acid of S219V is mutated from leucine (L) to phenylalanine (F), and the 138 th amino acid is mutated from isoleucine (I) to lysine (K);

12D: S219V, the 28 th amino acid is mutated from histidine (H) to leucine (L), the 111 th amino acid is mutated from leucine (L) to phenylalanine (F), and the 196 th amino acid is mutated from glutamic acid (Q) to histidine (H);

32C: leucine (L) is mutated into histidine (H) at the 111 th amino acid position of S219V, and serine (S) is mutated into glycine (G) at the 135 th amino acid position; the 187 th amino acid is mutated from methionine (M) to isoleucine (I).

FIG. 1 shows that TEVp mutant 12D has the highest enzyme cutting efficiency in 4M urea solution; FIG. 2 shows that TEVp mutant 4D has the highest enzyme cutting efficiency in 4M urea; FIG. 3 shows that TEVp mutant 32C has the highest enzyme digestion efficiency in 4M urea; FIGS. 4A and 4B show that TEVp variant 4D is the most efficient enzyme digestion in 4M urea at 25 ℃. When the concentration of urea exceeds 4M, the enzyme cutting efficiency of each mutant is gradually reduced.

Fig. 5 shows that TEVp mutants 4D, 12D, and 32C screened according to the present invention have significantly lower in vivo cleavage activity than the S219V control (based on 0 hour band comparison), and have slightly less in vitro cleavage activity than or equal to S219V.

Fig. 6 shows that the solubility of each TEVp mutant fusion protein was low in 0.5M urea (only a small amount of protein in the diluted supernatant, mostly in the diluted pellet), demonstrating that the TEVp mutant fusion protein was present in pellet form after dilution to 0.5M urea.

The TEV protease mutants used in the preparation of ACTH polypeptides of the invention are any of table 1. The TEV protease mutant screened by the invention can be used for preparing other recombinant polypeptides and proteins.

Example 2: preparation of ACTH Polypeptides with TEVP-ACTH fusion proteins

Construction of TEVP-ACTH fusion protein expression vector

In example 1, the carboxyl-terminal end of ACTH released by cleavage of the fusion protein carries a His tag, which is required to be removed in actual production. Therefore, it is necessary to introduce a stop codon downstream of the ACTH gene by PCR. The specific process is as follows: the plasmid with the TEV protease variant 12D, which performed best in step 3 of example 1, was used as a template, and the Avi-TEVP-sTEV-ACTH region in the open reading frame of the vector was amplified together by PCR (TEVP variant 12D sequence SEQ ID NO.5, ACTH gene sequence SEQ ID NO. 13). Amplification was performed with KOD-plus DNA high fidelity polymerase (tokyo) using the following procedure: pre-denaturation at 95 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, extension at 68 ℃ for 1min for 12s, amplification for 30 cycles), and introducing stop codon at the downstream of the gene. The primers are as follows:

TEV-ACTH-F:5-CCACCATGGCCGGTCTGAATGATATTTTTGAAGC-3

TEV-ACTH-R:5-

AGAGCGGCCGCTTATTAAAATTCCAGCGGAAATGCTTCTGC-3

the underlined parts are the restriction sites Nco1 and Not1, respectively. After the PCR product and the vector pET-28b (Novagen) were digested with Nco1 and Not1 at 37 ℃ for 3 hours, the digested fragments were recovered in gel, and then ligated with T4DNA ligase at 20 ℃ for 2 hours. The ligation products were transformed into DH 5. alpha. competent cells, the transformants were plated on kanamycin-resistant (50. mu.g/ml) LB plates and cultured until single colonies grew out, the single colonies were picked, the plasmids were extracted for restriction enzyme validation, and the recombinant plasmids were subjected to Kingsry sequencing to obtain the plasmid pET-28b-Avi-TEVP-sTEV-ACTH (where the sTEV sequence is in the TEVP-ACTH plasmid).

Induced expression, purification, enzyme digestion and verification of TEVP-ACTH fusion protein

The constructed expression vector is transformed. The specific process comprises the following steps: 50ng of plasmid pET-28b-TEVP-sTEV-ACTH was added to the corresponding chemocompetent cells Rosetta2(DE3), ice-cooled for 30 minutes, heat-shocked at 42 ℃ for 45 seconds, ice-cooled for 2 minutes, 1ml of antibiotic-free LB medium was added, and cultured at 37 ℃ for 1 hour, 100. mu.l of the bacterial solution was spread on LB plates containing kanamycin (50. mu.g/ml) + chloramphenicol (34. mu.g/ml), and cultured at 37 ℃ until single colonies grew out.

Inducible expression of TEVP-ACTH fusion protein: single colonies were picked and cultured in LB liquid medium containing kanamycin (50. mu.g/ml) and chloramphenicol (34. mu.g/ml) to an OD600 of 0.5 to 0.8. Inoculating the culture into LB liquid culture medium at a volume ratio of 1:50, culturing at 37 deg.C with vigorous shaking until OD600 is 0.5-0.8, adding IPTG with final concentration of 2mM, and inducing at 37 deg.C for 4 h. During expression, the TEVP-ACTH fusion protein exists as insoluble inclusion bodies. After the completion of fermentation, the collected cells were sonicated, washed with a washing buffer (composition: 50mM Tris-HCl, 200mM NaCl, 10mM EDTA, 10 mM. beta. -Mercaptoethanol, 0.5% Triton X-100) several times to obtain a crude extract of TEVP-ACTH fusion protein, and then the TEVP-ACTH fusion protein was dissolved in a buffer containing a denaturant, guanidine hydrochloride or Urea (composition: 50mM Tris-HCl pH8.0, 1mM EDTA,2mM DTT,8M Urea or 6M GuHCl) to prepare a denatured solution.

The fusion protein denatured solution was diluted ten times with TEVP digestion buffer (3.556M Urea, 50mM Tris-HCl,1mM EDTA,2mM DTT, pH8.0) to make the final concentration of Urea 4M, and self-digested overnight at 25 ℃, and after 8-fold dilution of the digested product (50mM Tris-HCl pH8.0 dilution), TEV protease and the fusion protein not digested precipitated due to poor solubility (see FIG. 6), while ACTH polypeptide could be dissolved in the solution, and thus the supernatant was collected after centrifugation at 13000g for 30 minutes at 4 ℃ and 13000g to obtain ACTH stock solution.

Purification and preservation of ACTH

Filtering impurities in ACTH stock solution by using a 0.22-micron filter membrane, carrying out ultrafiltration concentration by using a 1k ultrafiltration centrifugal tube (Millipore) and desalting at the same time, then precipitating by using 50% ammonium sulfate, forming suspended matters in the ACTH on the upper layer of the solution, carefully collecting, dissolving by using PBS, carrying out ultrafiltration concentration and desalting, thus obtaining high-purity recombinant human-source or pig-source ACTH solution, and freeze-drying and storing.

Structural characterization of ACTH

And (4) measuring the molecular weight of the purified ACTH by using a mass spectrometer, wherein the measured molecular weight value is consistent with a theoretical value. The results are shown in FIG. 10.

Activity assay of ACTH

Taking healthy 2-week-old SD rat, anesthetizing with 1% sodium pentobarbital (40mg/kg), taking out adrenal gland under aseptic condition, removing quilt and medulla, placing into Hanks balanced salt solution, and cutting into 1mm³The fragments with the size are transferred into a digestive solution containing collagenase type I and DNA enzyme for digestion for 1 hour, the fragments are shaken and mixed evenly at intervals of 5 minutes, the mechanical dispersed cells are blown and sucked by a pipette for a plurality of times to form suspension, the suspension is filtered into a 50ml centrifugal tube by a cell sieve, the centrifugation is carried out for 10 minutes at 1000Xg, the supernatant is carefully sucked off, the precipitated cells are washed for 2 times by Hanks liquid, and finally, the suspension is re-suspended by DMEM/F12 culture medium (Gibco) containing 20 percent fetal calf serum, and the concentration is adjusted to be 2X10⁵Each cell was inoculated into a 90mm dish and cultured at 37 ℃ under 5% CO 2. Observing the growth process and morphological change of the adrenal cells under an inverted phase contrast microscope, and after culturing for 48 hours, observing adherent growth of the adrenal cells under the microscope, wherein the cell volume is increased, the cytoplasm is circular or polygonal, the cytoplasm is large, the cytoplasm is transparent, and a plurality of particles with regular sizes are arranged in the cytoplasm, so that the rat adrenal cortex cells are obtained.

The purified ACTH was incubated with isolated rat adrenal cortex cells. Specifically, ACTH (cell-to-ACTH volume ratio 400:1) was added at different concentrations (0.1 μ U, 0.2 μ U, 2 μ U, 20 μ U, 200 μ U, 1U ═ 10 μ g protein) to each group of adrenal cells, and after incubation at 37 ℃ for 24 hours, the culture broth was taken, and after cell debris was removed by centrifugation, the corticosterone concentration was measured by elisa (see where "PCNA expression and its meaning in rat adrenal cell culture", journal of clinical urology 21.8(2006): 625-626), which showed that the corticosterone concentration gradually increased with the increase in ACTH concentration. ACTH stimulates cells to secrete steroid hormones, and the activity of ACTH is quantified by measuring the concentration of corticosterone produced (ELISA method). The results show that several batches of ACTH after purification have high bioactivity and activity stability. The results are shown in FIG. 11.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, so that any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention will still fall within the scope of the technical solution of the present invention without departing from the content of the technical solution of the present invention.

Sequence listing

SEQ ID NO.1 (wild type TEVP)

GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWGLNADSVLWGGHKVFMSKPEEPFQPVKEATQLMNELVYSQ

SEQ ID NO.2 (human ACTH amino acid sequence)

SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF

[ SEQ ID NO. 3: TEVP (219V) nucleotide sequence

GGAGAAAGCTTGTTTAAGGGACCACGTGATTACAACCCGATATCGAGCACCATTTGTCATTTGACGAATGAATCTGATGGGCACACAACATCGTTGTATGGTATTGGATTTGGTCCCTTCATCATTACAAACAAGCACTTGTTTAGAAGAAATAATGGAACACTGTTGGTCCAATCACTACATGGTGTATTCAAGGTCAAGAACACCACGACTTTGCAACAACACCTCATTGATGGGAGGGACATGATAATTATTCGCATGCCTAAGGATTTCCCACCATTTCCTCAAAAGCTGAAATTTAGAGAGCCACAAAGGGAAGAGCGCATATGTCTTGTGACAACCAACTTCCAAACTAAGAGCATGTCTAGCATGGTGTCAGACACTAGTTGCACATTCCCTTCATCTGATGGCATATTCTGGAAGCATTGGATTCAAACCAAGGATGGGCAGTGTGGCAGTCCATTAGTATCAACTAGAGATGGGTTCATTGTTGGTATACACTCAGCATCGAATTTCACCAACACAAACAATTATTTCACAAGCGTGCCGAAAAACTTCATGGAATTGTTGACAAATCAGGAGGCGCAGCAGTGGGTTAGTGGTTGGGGATTAAATGCTGACTCAGTATTGTGGGGGGGCCATAAAGTTTTCATGGTTAAACCTGAAGAGCCTTTTCAGCCAGTTAAGGAAGCGACTCAACTCATGAATGAATTGGTGTACTCGCAA

SEQ ID NO.4(4D TEVP)

GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICFVTTNFQTKSMSSMVSDTSCTFPSSDGKFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWGLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNELVYSQ

SEQ ID NO.5(12D TEVP)

GESLFKGPRDYNPISSTICHLTNESDGLTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICFVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAHQWVSGWGLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNELVYSQ

SEQ ID NO.6(32C TEVP)

GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICHVTTNFQTKSMSSMVSDTSCTFPSGDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFIELLTNQEAQQWVSGWGLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNELVYSQ

SEQ ID NO.7

ENLYFQS

SEQ ID NO.8

ENLYFQH

SEQ ID NO.9

EXXYXQ(G/S)

SEQ ID NO.10(S219V)

GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWGLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNELVYSQ

SEQ ID NO.11:EXXYXQS

SEQ ID NO. 12: sTEV (nucleotide sequence of TEVp cleavage site used in example)

GAAAATCTGTATTTTCAGAGC

ACTH DNA nucleotide sequence of SEQ ID NO.13

AGCTATAGCATGGAACATTTTCGTTGGGGTAAACCGGTTGGTAAAAAACGTCGTCCGGTTAAAGTTTATCCGAATGGTGCAGAAGATGAATCGGCAGAAGCATTTCCGCTGGAATTT

SEQ ID NO.14:TEV(4D)

GGAGAAAGCTTGTTTAAGGGACCACGTGATTACAACCCGATATCGAGCACCATTTGTCATTTGACGAATGAATCTGATGGGCACACAACATCGTTGTATGGTATTGGATTTGGTCCCTTCATCATTACAAACAAGCACTTGTTTAGAAGAAATAATGGAACACTGTTGGTCCAATCACTACATGGTGTATTCAAGGTCAAGAACACCACGACTTTGCAACAACACCTCATTGATGGGAGGGACATGATAATTATTCGCATGCCTAAGGATTTCCCACCATTTCCTCAAAAGCTGAAATTTAGAGAGCCACAAAGGGAAGAGCGCATATGTTTTGTGACAACCAACTTCCAAACTAAGAGCATGTCTAGCATGGTGTCAGACACTAGTTGCACATTCCCCTCATCTGATGGCAAATTCTGGAAGCATTGGATTCAAACCAAGGATGGGCAGTGTGGCAGTCCATTAGTATCAACTAGAGATGGGTTCATTGTTGGTATACACTCAGCATCGAATTTCACCAACACAAACAATTATTTCACAAGCGTGCCGAAAAACTTCATGGAATTGTTGACAAATCAGGAGGCGCAGCAGTGGGTTAGTGGTTGGGGATTAAATGCTGACTCAGTATTGTGGGGGGGCCATAAAGTTTTCATGGTTAAACCTGAAGAGCCTTTTCAGCCAGTTAAGGAAGCGACTCAACTCATGAATGAATTGGTGTACTCGCAA

SEQ ID NO.15:TEV(12D)

GGAGAAAGCTTGTTTAAGGGACCACGTGATTACAACCCGATATCGAGCACCATTTGTCATTTGACGAATGAATCTGATGGGCTCACAACATCGTTGTATGGTATTGGATTTGGTCCCTTCATCATTACAAACAAGCACTTGTTTAGAAGAAATAATGGAACACTGTTGGTCCAATCACTACATGGTGTATTCAAGGTCAAGAACACCACGACTTTGCAACAACACCTCATTGATGGGAGGGACATGATAATTATTCGCATGCCTAAGGATTTCCCACCATTTCCTCAAAAGCTGAAATTTAGAGAGCCACAAAGGGAAGAGCGCATATGTTTTGTGACAACCAACTTCCAAACTAAGAGCATGTCTAGCATGGTGTCAGACACTAGTTGCACATTCCCTTCATCTGATGGCATATTCTGGAAGCATTGGATTCAAACCAAGGATGGGCAGTGTGGCAGTCCATTAGTATCAACTAGAGATGGGTTCATTGTTGGTATACACTCAGCATCGAATTTCACCAACACAAACAATTATTTCACAAGCGTGCCGAAAAACTTCATGGAATTGTTGACAAATCAGGAGGCGCATCAGTGGGTTAGTGGTTGGGGATTAAATGCTGACTCAGTATTGTGGGGGGGCCATAAAGTTTTCATGGTTAAACCTGAAGAGCCTTTTCAGCCAGTTAAGGAAGCGACTCAACTCATGAATGAATTGGTGTACTCGCAA

SEQ ID NO.16:TEV(32C)

GGAGAAAGCTTGTTTAAGGGACCACGTGATTACAACCCGATATCGAGCACCATTTGTCATTTGACGAATGAATCTGATGGGCACACAACATCGTTGTATGGTATTGGATTTGGTCCCTTCATCATTACAAACAAGCACTTGTTTAGAAGAAATAATGGAACACTGTTGGTCCAATCACTACATGGTGTATTCAAGGTCAAGAACACCACGACTTTGCAACAACACCTCATTGATGGGAGGGACATGATAATTATTCGCATGCCTAAGGATTTCCCACCATTTCCTCAAAAGCTGAAATTTAGAGAGCCACAAAGGGAAGAGCGCATATGTCATGTGACAACCAACTTCCAAACTAAGAGCATGTCTAGCATGGTGTCAGACACTAGTTGCACATTCCCTTCAGGTGATGGCATATTCTGGAAGCATTGGATTCAAACCAAGGATGGGCAGTGTGGCAGTCCATTAGTATCAACTAGAGATGGGTTCATTGTTGGTATACACTCAGCATCGAATTTCACCAACACAAACAATTATTTCACTAGCGTGCCGAAAAACTTCATTGAATTGTTGACAAATCAGGAGGCGCAGCAGTGGGTTAGTGGTTGGGGATTAAATGCTGACTCAGTATTGTGGGGGGGCCATAAAGTTTTCATGGTTAAACCTGAAGAGCCTTTTCAGCCAGTTAAGGAAGCCACTCAACTCATGAATGAATTGGTGTACTCGCAA

Sequence listing

<110> Kangkeda Biotech Co., Ltd

<120> TEV protease variants, fusion proteins thereof, and methods of making and using

<130> C18P3301

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 242

<212> PRT

<213> tobacco etch virus

<400> 1

Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser

1 5 10 15

Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu

20 25 30

Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe

35 40 45

Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe

50 55 60

Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg

65 70 75 80

Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln

85 90 95

Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val

100 105 110

Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr

115 120 125

Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile

130 135 140

Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp

145 150 155 160

Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn

165 170 175

Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn

180 185 190

Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Gly Leu Asn Ala Asp Ser

195 200 205

Val Leu Trp Gly Gly His Lys Val Phe Met Ser Lys Pro Glu Glu Pro

210 215 220

Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr

225 230 235 240

Ser Gln

<210> 2

<211> 39

<212> PRT

<213> human

<400> 2

Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val Gly Lys Lys

1 5 10 15

Arg Arg Pro Val Lys Val Tyr Pro Asn Gly Ala Glu Asp Glu Ser Ala

20 25 30

Glu Ala Phe Pro Leu Glu Phe

35

<210> 3

<211> 726

<212> DNA

<213> tobacco etch virus

<400> 3

ggagaaagct tgtttaaggg accacgtgat tacaacccga tatcgagcac catttgtcat 60

ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg gtattggatt tggtcccttc 120

atcattacaa acaagcactt gtttagaaga aataatggaa cactgttggt ccaatcacta 180

catggtgtat tcaaggtcaa gaacaccacg actttgcaac aacacctcat tgatgggagg 240

gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa gctgaaattt 300

agagagccac aaagggaaga gcgcatatgt cttgtgacaa ccaacttcca aactaagagc 360

atgtctagca tggtgtcaga cactagttgc acattccctt catctgatgg catattctgg 420

aagcattgga ttcaaaccaa ggatgggcag tgtggcagtc cattagtatc aactagagat 480

gggttcattg ttggtataca ctcagcatcg aatttcacca acacaaacaa ttatttcaca 540

agcgtgccga aaaacttcat ggaattgttg acaaatcagg aggcgcagca gtgggttagt 600

ggttggggat taaatgctga ctcagtattg tgggggggcc ataaagtttt catggttaaa 660

cctgaagagc cttttcagcc agttaaggaa gcgactcaac tcatgaatga attggtgtac 720

tcgcaa 726

<210> 4

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> TEV protease mutant 4D

<400> 4

Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser

1 5 10 15

Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu

20 25 30

Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe

35 40 45

Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe

50 55 60

Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg

65 70 75 80

Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln

85 90 95

Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Phe Val

100 105 110

Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr

115 120 125

Ser Cys Thr Phe Pro Ser Ser Asp Gly Lys Phe Trp Lys His Trp Ile

130 135 140

Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp

145 150 155 160

Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn

165 170 175

Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn

180 185 190

Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Gly Leu Asn Ala Asp Ser

195 200 205

Val Leu Trp Gly Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro

210 215 220

Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr

225 230 235 240

Ser Gln

<210> 5

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> TEV protease mutant 12D

<400> 5

Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser

1 5 10 15

Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly Leu Thr Thr Ser Leu

20 25 30

Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe

35 40 45

Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe

50 55 60

Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg

65 70 75 80

Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln

85 90 95

Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Phe Val

100 105 110

Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr

115 120 125

Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile

130 135 140

Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp

145 150 155 160

Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn

165 170 175

Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn

180 185 190

Gln Glu Ala His Gln Trp Val Ser Gly Trp Gly Leu Asn Ala Asp Ser

195 200 205

Val Leu Trp Gly Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro

210 215 220

Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr

225 230 235 240

Ser Gln

<210> 6

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> TEV protease mutant 32C

<400> 6

Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser

1 5 10 15

Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu

20 25 30

Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe

35 40 45

Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe

50 55 60

Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg

65 70 75 80

Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln

85 90 95

Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys His Val

100 105 110

Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr

115 120 125

Ser Cys Thr Phe Pro Ser Gly Asp Gly Ile Phe Trp Lys His Trp Ile

130 135 140

Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp

145 150 155 160

Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn

165 170 175

Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Ile Glu Leu Leu Thr Asn

180 185 190

Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Gly Leu Asn Ala Asp Ser

195 200 205

Val Leu Trp Gly Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro

210 215 220

Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr

225 230 235 240

Ser Gln

<210> 7

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TEV protease cleavage site

<400> 7

Glu Asn Leu Tyr Phe Gln Ser

1 5

<210> 8

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TEV protease cleavage site

<400> 8

Glu Asn Leu Tyr Phe Gln His

1 5

<210> 9

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TEV protease cleavage site

<220>

<221> misc_feature

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 9

Glu Xaa Xaa Tyr Xaa Gln Gly

1 5

<210> 10

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> TEV protease mutant S219V

<400> 10

Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser

1 5 10 15

Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu

20 25 30

Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe

35 40 45

Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe

50 55 60

Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg

65 70 75 80

Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln

85 90 95

Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val

100 105 110

Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr

115 120 125

Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile

130 135 140

Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp

145 150 155 160

Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn

165 170 175

Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn

180 185 190

Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Gly Leu Asn Ala Asp Ser

195 200 205

Val Leu Trp Gly Gly His Lys Val Phe Met Val Lys Pro Glu Glu Pro

210 215 220

Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr

225 230 235 240

Ser Gln

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TEV protease cleavage site

<220>

<221> misc_feature

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<400> 11

Glu Xaa Xaa Tyr Xaa Gln Ser

1 5

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> TEVp cleavage site

<400> 12

gaaaatctgt attttcagag c 21

<210> 13

<211> 117

<212> DNA

<213> human

<400> 13

agctatagca tggaacattt tcgttggggt aaaccggttg gtaaaaaacg tcgtccggtt 60

aaagtttatc cgaatggtgc agaagatgaa tcggcagaag catttccgct ggaattt 117

<210> 14

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> TEV protease mutant 4D

<400> 14

ggagaaagct tgtttaaggg accacgtgat tacaacccga tatcgagcac catttgtcat 60

ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg gtattggatt tggtcccttc 120

atcattacaa acaagcactt gtttagaaga aataatggaa cactgttggt ccaatcacta 180

catggtgtat tcaaggtcaa gaacaccacg actttgcaac aacacctcat tgatgggagg 240

gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa gctgaaattt 300

agagagccac aaagggaaga gcgcatatgt tttgtgacaa ccaacttcca aactaagagc 360

atgtctagca tggtgtcaga cactagttgc acattcccct catctgatgg caaattctgg 420

aagcattgga ttcaaaccaa ggatgggcag tgtggcagtc cattagtatc aactagagat 480

gggttcattg ttggtataca ctcagcatcg aatttcacca acacaaacaa ttatttcaca 540

agcgtgccga aaaacttcat ggaattgttg acaaatcagg aggcgcagca gtgggttagt 600

ggttggggat taaatgctga ctcagtattg tgggggggcc ataaagtttt catggttaaa 660

cctgaagagc cttttcagcc agttaaggaa gcgactcaac tcatgaatga attggtgtac 720

tcgcaa 726

<210> 15

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> TEV protease mutant 12D

<400> 15

ggagaaagct tgtttaaggg accacgtgat tacaacccga tatcgagcac catttgtcat 60

ttgacgaatg aatctgatgg gctcacaaca tcgttgtatg gtattggatt tggtcccttc 120

atcattacaa acaagcactt gtttagaaga aataatggaa cactgttggt ccaatcacta 180

catggtgtat tcaaggtcaa gaacaccacg actttgcaac aacacctcat tgatgggagg 240

gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa gctgaaattt 300

agagagccac aaagggaaga gcgcatatgt tttgtgacaa ccaacttcca aactaagagc 360

atgtctagca tggtgtcaga cactagttgc acattccctt catctgatgg catattctgg 420

aagcattgga ttcaaaccaa ggatgggcag tgtggcagtc cattagtatc aactagagat 480

gggttcattg ttggtataca ctcagcatcg aatttcacca acacaaacaa ttatttcaca 540

agcgtgccga aaaacttcat ggaattgttg acaaatcagg aggcgcatca gtgggttagt 600

ggttggggat taaatgctga ctcagtattg tgggggggcc ataaagtttt catggttaaa 660

cctgaagagc cttttcagcc agttaaggaa gcgactcaac tcatgaatga attggtgtac 720

tcgcaa 726

<210> 16

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> TEV protease mutant 32C

<400> 16

ggagaaagct tgtttaaggg accacgtgat tacaacccga tatcgagcac catttgtcat 60

ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg gtattggatt tggtcccttc 120

atcattacaa acaagcactt gtttagaaga aataatggaa cactgttggt ccaatcacta 180

catggtgtat tcaaggtcaa gaacaccacg actttgcaac aacacctcat tgatgggagg 240

gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa gctgaaattt 300

agagagccac aaagggaaga gcgcatatgt catgtgacaa ccaacttcca aactaagagc 360

atgtctagca tggtgtcaga cactagttgc acattccctt caggtgatgg catattctgg 420

aagcattgga ttcaaaccaa ggatgggcag tgtggcagtc cattagtatc aactagagat 480

gggttcattg ttggtataca ctcagcatcg aatttcacca acacaaacaa ttatttcact 540

agcgtgccga aaaacttcat tgaattgttg acaaatcagg aggcgcagca gtgggttagt 600

ggttggggat taaatgctga ctcagtattg tgggggggcc ataaagtttt catggttaaa 660

cctgaagagc cttttcagcc agttaaggaa gccactcaac tcatgaatga attggtgtac 720

tcgcaa 726

Claims (42)

1. A TEV protease variant consisting of the amino acid sequence shown in SEQ ID No. 4, 5 or 6.
2. 2. A fusion protein comprising the TEV protease variant of claim 1.
3. 3. The fusion protein of claim 2, comprising the structure TEVp-sTEV-Y1,

   wherein Y1 is a polypeptide of interest;

   TEVp is the TEV protease variant of claim 1;

   sTEV is a TEV protease cleavage site, which is EXXYXQG/S/H, where X is any amino acid residue.
4. 4. The fusion protein of claim 3, wherein the cleavage site is selected from the group consisting of SEQ ID NOs 7 and 8.
5. 5. The fusion protein according to claim 3 or 4, wherein the polypeptide of interest is selected from ACTH, GLP-1/GLP-2, IFN- α, IFN- γ, Histatin, CCL5, SDF-1 α, IGF-1, Leptin, BNP or Ex-4.
6. 6. The fusion protein of claim 5, wherein the polypeptide of interest is ACTH.
7. 7. The fusion protein of claim 6, wherein ACTH is human ACTH.
8. 8. The fusion protein of claim 5, wherein ACTH is human ACTH having the amino acid sequence set forth in SEQ ID No. 2.
9. 9. The fusion protein of any one of claims 3-8, wherein the TEVp is directly linked to the sTEV or separated by one or more amino acid residues, provided that the TEVp can recognize and cleave the sTEV.
10. 10. The fusion protein of any one of claims 3-8, wherein sTEV is directly linked to Y1 or separated by one or more amino acid residues, provided that TEVp recognizes and cleaves sTEV.
11. 11. The fusion protein of claim 10, wherein sTEV is directly linked to Y1.
12. 12. The fusion protein of any one of claims 2-8, further comprising a tag.
13. 13. The fusion protein of claim 12, wherein the tag is a purification tag.
14. 14. The fusion protein of claim 12, wherein the tag is selected from the group consisting of: a His tag, a Maltose Binding Protein (MBP) tag, a glutathione transferase (GST) tag, a NusA tag, a SUMO tag, an Avi tag, a T7 tag, an S tag, a Flag tag, an HA tag, a c-myc tag, or a Strep II tag.
15. 15. The fusion protein of claim 12, wherein the tag is at the N-terminus of the fusion protein.
16. 16. The fusion protein according to claim 3, wherein the N-terminus of the TEVp is unlabeled.
17. 17. A polynucleotide encoding the TEV protease variant according to claim 1 or the fusion protein according to any one of claims 2-16.
18. 18. The polynucleotide of claim 17, selected from SEQ ID nos. 14-16.
19. 19. A polynucleotide construct comprising the polynucleotide of claim 17 or 18.
20. 20. An expression vector comprising the polynucleotide sequence of claim 17 or 18 or the polynucleotide construct of claim 19.
21. 21. The expression vector of claim 20, wherein the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.
22. 22. The expression vector of claim 21, which is a eukaryotic expression vector.
23. 23. The expression vector of claim 22, selected from the group consisting of: pRS314, pYES2, baculovirus-S2 expression system and pcDNA3.1.
24. 24. The expression vector of claim 21, which is a prokaryotic expression vector.
25. 25. The expression vector of claim 24, selected from the group consisting of: pET series expression vector, pQE series expression vector and pBAD series expression vector.
26. 26. A cell comprising the polynucleotide of claim 17 or 18, the polynucleotide construct of claim 19, or the expression vector of any one of claims 20-25.
27. 27. The cell of claim 26, which is a eukaryotic cell or a prokaryotic cell.
28. 28. The cell of claim 27, which is a eukaryotic cell selected from the group consisting of: saccharomyces cerevisiae, insect cell expression system.
29. 29. The cell of claim 27, which is a prokaryotic cell selected from the group consisting of: BL21, BL21(DE3), BL21(DE3) pLysS, Rosetta2, Rosetta2pLysS, Tuner (DE3), or Origami 2.
30. 30. A method of making a TEV protease variant according to claim 1, comprising:

    (1) culturing the cell of any one of claims 26-29 in a culture medium under conditions suitable for culturing the cell;

    (2) harvesting the culture medium, or lysing the cells to harvest the lysate;

    (3) purifying to obtain the TEV protease variant.
31. 31. Use of a TEV protease variant according to claim 1 for the preparation of a polypeptide of interest, wherein said TEV protease variant is expressed as a fusion protein with said polypeptide of interest.
32. 32. The use of claim 31, wherein the fusion protein is a fusion protein according to any one of claims 3-16.
33. 33. A method of making a polypeptide of interest, comprising:

    (1) culturing the cell of any one of claims 26-29 in a culture medium under conditions suitable for culturing the cell;

    (2) obtaining inclusion bodies of the fusion protein;

    (3) solubilizing the inclusion bodies under highly denaturing conditions of 8M urea or 6M guanidine hydrochloride;

    (4) incubating at 20-40 ℃ for a period of time under moderate to high denaturing conditions, said moderate to high denaturing conditions being 3M-5M urea or 1M-2M guanidine hydrochloride;

    (5) diluting with buffer solution and precipitating TEV protease;

    (6) removing the TEV protease precipitate to obtain the polypeptide of interest; and is

    (7) Purifying the polypeptide of interest.
34. 34. The method of claim 33, wherein the moderate to high denaturing conditions are 3.5M to 4.5M urea or 1.5M guanidine hydrochloride.
35. 35. The method of claim 34, wherein the high degree of denaturing conditions is 4M urea.
36. 36. The method of claim 33, wherein step (4) is performed at 25 ℃.
37. 37. The method of claim 33, wherein the time is 10-24 hours.
38. 38. The method of claim 37, wherein the time is 12 hours.
39. 39. The method of claim 33, wherein the buffer is Tris-HCl.
40. 40. The method of claim 33, wherein the buffer is 50mM Tris-HCl.
41. 41. The method of claim 33, wherein TEV protease precipitate is removed by centrifugation.
42. 42. The method according to claim 33, wherein said purification is performed with a technique selected from the group consisting of: salting out, ultrafiltration, organic solvent precipitation, gel filtration, ion exchange chromatography column, and reversed phase high performance liquid chromatography.

Images