RU2619217C1 - Temperaturically intelligent mutant intein for the insoluble expression of the precursor of the target protein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: temperature-sensitive mutant PRP8 mini-intein from Penicillium chrysogenum insoluble for expression in Escherichia coli cells of the target protein precursor is provided. Mutant PRP8 mini-intein comprises a substitution of an amino acid residue leucine at position 93 to a proline residue, substitution of a cysteine residue at position 1 to an alanine residue and optionally contains a substitution of a cysteine residue at position 11 to tyrosine residue in the original sequence PRP8 mini-intein from Penicillium chrysogenum, consisting of 157 amino acid residues. When using a mutant intein combined and insoluble expression self-splitting ofpurified fusion protein from the refolding process that does not require use of proteases. The degree of processing for the modeling of SUMO target protein is more than 50%, and for neurotoxin CVIE - about 60%.

EFFECT: improved properties.

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Description

We will call insoluble expression such an expression variant of the target protein, in which the target expressed product is isolated from cells in the fraction of insoluble cellular proteins precipitated by centrifugation.

The present invention relates to the field of biotechnology and relates to directed insoluble expression of the target protein obtained as part of a hybrid precursor (fusion) comprising a mutant variant of the PRP8 intein from Penicillium chrysogenum, followed by sequential denaturation and renaturation processes, during which the mutant intein is cleaved and target protein release.

Heterologous expression of the protein in Escherichia coli cells often leads to the accumulation of the target protein in an insoluble form (in the form of “inclusion bodies”). In most cases, this is an undesirable fact, since it requires additional steps for refolding the protein of interest to the researcher. However, in some cases, when expressing small peptides that rapidly degrade in the cell or toxic proteins, insoluble expression can serve as a simple and effective way to obtain them [1].

Along with the proteins used as modules for the construction of fusion in order to increase the solubility of the target protein (thioredoxin, glutathione transferase (GST), SUMO [1]), the use of protein modules is common, expression in fusion with which leads to aggregation and accumulation of protein in insoluble form. These modules find their application in the production of small peptides and are presented in a number of commercial offers. The most popular are the ketosteroid isomerase (13.5 kDa), the TAF12 domain, etc. [2].

The advantage of the “insoluble” modules is to simplify the process of purification of the target protein: the easily separated insoluble component of lysed cells is mostly represented by the protein components of inclusion bodies.

A frequently used approach to the release of the target protein from the fusion composition is the placement of a protease recognition site at the boundary of the target protein and the protein module (for example, enterokinase site, KEX2 endopeptidase site, and others). Protease specifically cuts fusion, releasing the target protein. Such a process requires additional protease costs and time, which may become critical for the economic feasibility of production in the industrial production of the target protein [3].

Deprived of these disadvantages is an approach based on the use of self-cleaving proteins. Discovered in 1990, inteins are protein elements that can autocatalytically self-cleave and / or cleave at the border of their terminal amino acids. This property of them has been successfully implemented in commercial vectors IMPACT® System and IMPACT-CN® System (New England Biolabs) [4]. Protein cleavage is accompanied by the formation of a complete peptide bond between amino acids flanking intein. Mutation of conserved N'- and C'-terminal amino acids leads to the impossibility of complete protein splicing, while the activity of cleavage at the N- or C-terminal amino acids is preserved. Thus, the replacement of the first cysteine with alanine imposes a ban on the termination of the bond at the N-terminus of intein [5]. Such an intein acquires the properties of a self-cleaving label.

Initially, all inteins contained an endonuclease domain responsible for integration of the intein gene (the size of such inteins is ~ 400-500 bp), which is not necessary for splicing [5]. Inteins that do not have an endonuclease domain, the so-called mini-inteins, are preferable for biotechnology due to their smaller size.

Among the known features of inteins that limit their use for the preparation of target protein products are usually the dependence of the efficiency of C-terminal processing on the nature of the amino acid residues following the processing site [20]. In addition, it is known from the prior art that, in general, the splicing of a protein, even if it is completely catalyzed by intein, can be significantly affected by the sequences of exterins (flanking regions of the protein), and some host proteins may be incompatible with the activity of inteins, i.e. cannot be obtained in a biologically active state ([21], description p. 5 line 5 - p. 6 line 5 with reference to [22]; in particular, p. 460 para. 2, as well as [23], in particular with .855 middle and right column).

The first restriction is general in nature and applies to all used inteins, including the claimed one. The second limitation does not apply to the claimed invention, in which the expression of the target protein in the composition of the precursor in a biologically active state is not a prerequisite, since the conversion of the processed target protein to the active state is carried out at subsequent stages in the process of renaturation of the purified protein and is achieved by means known from the level technicians.

The above commercially available IMPACT® System and IMPACT-CN® System (NEB) vectors are designed for soluble fusion expression. In variants of these systems, in which intein is located at the N-end of the fusion, the processing speed is controlled by pH and temperature: with increasing temperature and / or increasing the cultivation time, the proportion of uncontrollably processed protein increases, which reduces the yield of the final product [6].

An expression system based on insoluble fusion expression is devoid of this drawback. In particular, a system based on the autocatalytic protease N ^pro (168 a.a.) from a virus of the genus Pestivirus is known [7, 8]. We will consider this protease as the closest analogue of the claimed mutant intein.

Protease N ^{pro is} autocatalytically cleaved from fusion at the border of its C-terminal amino acid [9]. When using N ^pro, two phenomena are combined: insoluble expression (this fusion is mainly in the insoluble fraction of the cell) and self-removal of N ^pro from fusion in the process of renaturation of the purified protein, which does not require the use of proteases.

An object of the present invention is to expand the arsenal of self-cleaving agents for insoluble expression of a target protein precursor.

The problem was solved by obtaining a temperature-sensitive mutant intein for insoluble expression of the target protein precursor in Escherichia coli cells, corresponding to the intein amino acid sequence belonging to the PRP8 family, containing the replacement of the amino acid residue of leucine at position 93 with the proline residue, the replacement of the cysteine residue in position 1 with the alanine residue and not necessarily containing a substitution of cysteine at position 11 for a tyrosine residue.

Such a mutant intein is incapable of correct folding at elevated (nonpermissive) temperature, which leads to insoluble expression [10]. To obtain a temperature-sensitive intein, intein was selected from Penicillium chrysogenum [11, 12].

A widely known method of obtaining temperature-sensitive protein mutants is based on screening a library of mutational variants obtained by random mutagenesis using PCR [10]. This approach was used by us in the present work, as a result of which a variant of intein with the Leu93Pro mutation was selected, which is capable of predominantly insoluble expression in E. coli at a temperature of 37 ° C.

The present invention is based on the use of mutant intein from the body of P. chrysogenum (157 amino acid residues) [11] and is an independently developed approach similar to the N ^pro system. When using the claimed mutant intein, as well as when using the N ^pro protease, two phenomena are combined: insoluble expression and self-elimination from fusion in the process of renaturation of the purified protein that does not require the use of proteases.

The inventive intein contains the following mutations:

1) Cys1Ala blocking cleavage at the N-terminus of intein. At the same time, the ability of intein to autocatalytically cleave along the boundary between its last amino acid (Asnl57) and the next amino acid of the protein fused with intein is retained;

2) Leu93Pro (first obtained by us), which changes the intracellular expression of the fusions formed by it in E. coli cells in comparison with intein that does not contain this mutation: at expression at a temperature of 37 ° C, fusion with intein containing this mutation mainly accumulate in fractions insoluble cellular proteins in unprocessed form. Upon subsequent renaturation at a low temperature (4 ° C), the mutant intein is efficiently cleaved.

3) Additionally, the claimed intein contains a Cys11Tyr mutation, which is optional.

The invention is illustrated by the following drawings.

FIG. 1. Scheme of the plasmid pET28b_ (His) × 10_IntMUT.

FIG. 2. Electrophoregram for the expression of SUMO fusion with mutant intein (Cys1Ala, Leu93Pro, Cys11Tyr) and control variant of intein (Cys1Ala, Cys11Tyr): Lanes: 1 - marker, 2-4: [total protein ⎜ supernatant ⎜ sediment] of cells expressing

(His) × 10_IntMUT_SUMO, 5-7: [total protein ⎜ supernatant ⎜ sediment] cells expressing (His) × 10_IntWT_SUMO.

FIG. 3. Electrophoregram of the processed protein (His) × 10_IntMUT_SUMO. Lanes: 1 — marker, 2 — total protein, 3 — precipitate dissolved in 8M urea, 4 — renatured protein in 1M urea after processing.

FIG. 4. Electrophoregram of GFP fusion expression with mutant intein (Cys1Ala, Leu93Pro, Cys11Tyr) and a control variant of intein (Cys1Ala, Cys11Tyr). Lanes: 1 - marker, 2-4: [total protein ⎜ supernatant ⎜ sediment] of cells expressing (His) × 10_IntMUT_GFP, 5-7: [total protein ⎜ supernatant ⎜ sediment] of cells expressing (His) × 10_IntWT_GFP.

FIG. 5. Electrophoregram of the processed protein (His) × 10_IntMUT_GFP. Lanes: 1 — marker, 2 — total protein, 3 — precipitate dissolved in 8M urea, 4 — protein renatured in 1M urea after processing.

FIG. 6. Electrophoregram of protein expression (His) × 10_IntMUT and (His) × 10_IntWT. Lanes: 1 - marker, 2-4: [total protein ⎜ supernatant ⎜ sediment] cells expressing (His) × 10_IntMUT, 5-7: [total protein ⎜ supernatant ⎜ sediment] cells expressing (His) × 10_IntWT.

FIG. 7. Electrophoregram for expression of fusion IFN with mutant intein (Cys1Ala, Leu93Pro, Cys11Tyr) and a control variant of intein (Cys1Ala, Cys11Tyr). Lanes: M - marker (kDa), 1-3: [total protein ⎜ supernatant ⎜ sediment] of cells expressing (His) × 10_IntMUT_IFN, 4-6: [total protein ⎜ supernatant ⎜ sediment] of cells expressing (His) × 10_IntWT_IFN .

FIG. 8. Electrophoregram of expression of fusion Ssm6A with mutant intein (Cys1Ala, Leu93Pro, Cys11Tyr). Lanes: M - marker (kDa), 1-3: [total protein ⎜ supernatant ⎜ sediment] cells expressing (His) × 10_IntMUT_Ssm6A, 4 - protein precipitate (His) × 10_IntMUT_Ssm6A dissolved in 8M urea, 5 - renatured in 1M urea protein after processing.

FIG. 9. Electrophoregram of expression of fusion CVIE with mutant intein (Cys1Ala, Leu93Pro, Cys11Tyr). Lanes: M - marker (kDa), 1-3: [total protein ⎜ supernatant ⎜ sediment] cells expressing (His) × 10_IntMUT_CVIE, 4 - protein precipitate (His) × 10_IntMUT_CVIE dissolved in 8M urea, 5 - renatured in 1M urea protein after processing.

Example 1. Obtaining the plasmid pET28b_ (His) × 10_IntWT containing the intein gene modified at the N-terminus (Cys1Ala, Cys11Tyr) from the Penicillium chrysogenum genome (control)

The intein gene carrying two mutations Cys1Ala and Cys11Tyr and not carrying the key mutation (Leu93Pro) was amplified in PCR with total P. chrysogenum genomic DNA using N1 + N2 primers

Primers are designed based on the presented intein sequence in the GeneBank database (AM042015.1) with the calculation of the insertion of the declared mutations and flanking restriction gene sites for cloning. The total genomic DNA of P. chrysogenum, isolated by the standard method [13] from the P. chrysogenum VKPM F-3 strain, is used as a matrix.

The obtained PCR fragment (~ 500 pairs of nucleotides in size) was cloned into the pET28b plasmid (Novagen) at the NcoI and XhoI restriction sites, and the plasmid pET28b_ (His) × 10_IntWT was obtained. Gene expression in the resulting vector is regulated by the T7 promoter [14].

For cloning and subsequent expression in accordance with standard methods [15], E. coli XL1blue and BL21 (DE3) strains were used.

Example 2. Obtaining the plasmid pET28b_ (His) × 10_IntMUT containing the mutant intein gene (Cys1Ala, Cys11Tyr, Leu93Pro)

The intein gene carrying three mutations Cys1Ala, Cys11Tyr and Leu93Pro is obtained by PCR in 2 rounds using primers N1 and N2 and additional primers N3 and N4 having the following structure:

The introduced nucleotide substitution (primers N3, N4) leading to the Leu93Pro mutation is highlighted in bold (ctt → cct).

In the first round, two PCR reactions are carried out using primer pairs N1 + N4 and N3 + N2. The matrix used is the plasmid pET28b_ (His) × 10_IntWT, the preparation of which is described in Example 1. In the second round, primers N1 and N2 are used, and two PCR reaction products obtained in the first round are used as a matrix.

The obtained PCR fragment (SEQ ID NO 1) was cloned into the pET28b plasmid at the NcoI and XhoI restriction sites to obtain the plasmid pET28b_ (His) × 10_IntMUT (Fig. 1).

Thus, the pET28b_ (His) × 10_IntMUT plasmid is obtained containing the mutant intein gene (Cys1Ala, Cys11Tyr, Leu93Pro) with the Ser-Gly-Ser three amino acid linker added to the 3'-end (in front of the stop codon), the coding sequence of which contains the recognition site BamHI restriction endonucleases for the convenience of subsequent cloning.

Example 3. Comparison of the expression of proteins (His) × 10_IntMUT and (His) × 10_IntWT

To demonstrate the difference in the properties of the intein carrying the Leu93Pro mutation from the intein of this non-carrying mutation, their intracellular expression is compared. Expression is carried out in cells of strain BL21 (DE3) transformed with the corresponding plasmids. Analysis of expressed proteins is carried out as described in Example 4.

The expressed protein (His) × 10_IntMUT accumulates mainly in the fraction of insoluble proteins, and the protein (His) × 10_IntWT accumulates mainly in the form of a soluble protein (Fig. 6).

Example 4. Expression and processing in vitro protein (His) × 10_IntMUT_SUMO using mutant intein

4-1. Expression

To demonstrate the ability of the claimed mutant intein to direct expression to inclusion bodies, the SUMO protein (Small Ubiquitin-like Modifier) is used as the target protein, which is used in biotechnology to create fusion with poorly soluble proteins to increase their solubility [1].

Compare the expression of SUMO in the composition of fusion with intein that carries the Leu93Pro mutation and intein that does not carry this mutation (control).

Plasmids containing genetic constructs encoding the fusion proteins (His) × 10_IntMUT_SUMO and (His) × 10_IntWT_SUMO (control) are obtained by cloning the plasmids described above (Example 1 and 2) of the SMT3 gene encoding the SUMO protein from Saccharomyces cerevisiae yeast at restriction sites I and XhoI amplified by PCR using appropriate primers carrying the restriction sites necessary for cloning [16].

Expression of fusion (His) × 10_IntMUT_SUMO and (His) × 10_IntWT_SUMO was analyzed in cells of E. coli strain BL21 (DE3).

Competent cells [17] of strain BL21 (DE3) are transformed with plasmids pET28b_ (His) × 10_IntMUT_SUMO or pET28b_ (His) × 10_IntWT_SUMO and plated on a LB medium of the following composition in wt. %: bacto-tryptone - 1, yeast extract - 0.5, sodium chloride - 1, agar 1.5, water - the rest containing a selective antibiotic kanamycin in an amount of 30 mg / l, and incubated for 12 hours at a temperature of 37 ° FROM. Colonies of grown transformants are seeded in LB liquid medium (50 ml of medium in a 1 L conical flask) with kanamycin and lactose (0.2% v / v) added to the medium to induce the expression of genetic constructs. Inoculated flasks were incubated for 14 hours at 37 ° C with shaking (250 rpm). The grown cells are precipitated by centrifugation (5000 rpm, 5 minutes) and destroyed in a French press [18].

The obtained cell lysates are separated into soluble and insoluble fractions by centrifugation (4 ° C, 13000 rpm, 15 minutes). The effective accumulation of the fusion protein precursor (His) × 10_IntMUT_SUMO in insoluble and unprocessed form was confirmed by protein gel electrophoresis. In FIG. Figure 2 shows the accumulation in the insoluble fraction of the unprocessed protein (His) × 10_IntMUT_SUMO (which corresponds to a molecular weight of 32 kDa). At the same time, the protein (His) × 10_IntWT_SUMO is not detected in the unprocessed state, its parts are observed in the lysate, which are the products of its processing SUMO and (His) × 10_IntWT.

4-2. Processing of the hybrid protein precursor (His) × 10_IntMUT_SUMO in vitro

Subsequent operations are carried out in ice.

After separation by centrifugation (13000 rpm, 10 minutes, 4 ° C) of insoluble components of the destroyed cells, the protein is denatured and renaturated from these insoluble components, during which autocatalytic cleavage of intein occurs. For this, the obtained precipitate from cell lysates is dissolved by adding to it a solution of 8M urea (~ 100 mg of precipitate in 2 ml of 8M urea) for 1 hour. The undissolved components are separated by centrifugation (13000 rpm, 10 min, 4 ° C). The content of the expressed hybrid protein in the supernatant is determined by electrophoretic method. For autocatalytic cleavage of the intein part, the resulting solution was diluted with water to a urea concentration of 1M, after which the samples were incubated at

temperature of 4 ° C for 3 days. The degree of protein processing determined by protein electrophoresis (Fig. 3) is more than 50%.

Example 5. Expression and processing of in vitro protein (His) × 10_IntMUT_GFP

To confirm the effectiveness of the claimed intein to carry out insoluble expression of the precursor protein, the GFP protein is used as a model protein [19]. The GFP gene is amplified in PCR from pEGFP (Clontech) plasmid and cloned into p ET28b_ (His) × 10_IntMUT and pET28b_ (His) × 10_IntWT plasmids, resulting in genetic constructs encoding the (His) × 10IntMUT_GFP and (His) × 10_IntMUT_ proteins , which are similar in structure to fusions with the SUMO protein (Example 4), between the last encoded amino acid intein (Asn) and the first GFP (Met) is placed the tri-amino linker Ser-Gly-Ser.

Obtaining and culturing strains containing these constructs, and analysis of the expressed proteins is carried out according to the method described in Example 4.

In FIG. Figure 4 shows the accumulation in the insoluble fraction of the unprocessed His × 10_IntMUT_GFP protein (which corresponds to a molecular weight of 43 kDa), which is not observed for His × 10_IntWT _GFP protein.

Further operations to clean inclusion bodies containing fusion (His) × 10_intMUT_GFP, as well as its processing, are carried out according to the procedure described in Example 4 for protein (His) × 10_IntMUT_SUMO.

From the results shown in FIG. 5, it is seen that the proportion of the processed by the method of protein electrophoresis. protein (His) × 10_IntMUT_GFP is less than the fraction of processed protein (His) × 10_IntMUT_SUMO (Fig. 3), which illustrates the dependence of the processing efficiency of the precursor protein on the properties of the target protein.

Example 6. Expression and processing in vitro protein (His) × 10_IntMUT_IFN

The effectiveness of the claimed intein for insoluble expression of the protein precursor is confirmed by the synthesis of the model target protein of human interferon alpha 2b (IFN) [24].

The IFN gene is obtained as a BamHI / XhoI DNA fragment from the plasmid pUC18 × - (Ser) IFN [24] and cloned into plasmids p ET28b_ (His) × 10_IntMUT or pET28b_ (His) × 10_IntWT, resulting in protein constructs encoding proteins (His) × 10_IntMUT_IFN or (His) × 10IntWT_IFN. In their composition, as well as in the fusion constructs with the SUMO protein (Example 4), the three amino acid linker Ser-Gly-Ser is located between the last amino acid residue of intein (Asn) and the first residue of mature IFN (Cys).

The cultivation of strains containing these constructs, and analysis of the expressed proteins is carried out according to the procedure described in Example 4.

In FIG. Figure 7 shows the accumulation in the insoluble fraction of exclusively unprocessed His × 10_IntMUT_IFN protein with a molecular weight of about 43 kD, while the expression of His × 10_IntWT_IFN construct shows that IFN protein with a molecular weight of about 18 kD is the main accumulated product.

Further operations to clean inclusion bodies containing fusion (His) × 10_intMUT_IFN, as well as its processing, require the use of specific specific conditions for their implementation, different from the conditions described in Example 4 for protein (His) × 10_IntMUT_SUMO. This illustrates the dependence of the properties of the resulting fusion and the efficiency of its processing on the properties of the target protein.

Example 7. Expression and processing of in vitro protein (His) × 10_IntMUT_Ssm6A

The effectiveness of the claimed intein for the insoluble expression of the protein precursor is confirmed by the synthesis of another model target protein - the recombinant neurotoxin Ssm6A from the poison of the Chinese red-headed centipede (Chinese red-headed centipede) Scolopendra subspinipes mutilans, the amino acid sequence and structure of which is disclosed in [25] .

For this purpose, a recombinant plasmid pET28b_ (His) × 10_IntMUT_Ssm6A containing a complex DNA fragment encoding fusion (His) × 10_IntMUT_Ssm6A was constructed.

The complex DNA fragment is obtained in two stages.

At the first stage, two overlapping DNA fragments are constructed:

• Fragment 1, which contains a DNA sequence containing a unique NcoI site at the 5'-end and encoding a protein (His) × 10_IntMUT, is obtained by PCR amplification using the plasmid pET28b_ (His) × 10_IntMUT as a DNA template. For amplification using the following pair of primers:

• Fragment 2, comprising a DNA sequence containing a unique XhoI site at the 3'-terminal portion and encoding the Ssm6A protein, is synthesized as a double-stranded DNA sequence during PCR using the following set of overlapping oligonucleotides:

The amplified DNA fragments were purified on an agarose gel using a Qiagen whale (Qiagen, cat. No. 28706) and used in a second step to prepare a complex DNA fragment.

At this stage, a mixture of purified overlapping fragments 1 and 2 is introduced into the PCR reaction mixture. N (his) and N (terl) are used as amplification primers. As a result of PCR, fragments 1 and 2 are ligated and a complex amplified DNA fragment of about 680 bp is obtained, encoding fusion (His) × 10_IntMUT_Ssm6A.

The resulting DNA fragment was purified on an agarose gel, digested with restriction enzymes NcoI and XhoI and cloned in the expression vector pET28b (Novagen) at the restriction sites NcoI and XhoI.

Cloning yields the plasmid pET28b_ (His) × 10_IntMUT_Ssm6A, which contains the fusion gene (His) × 10_IntMUT_Ssm6A under the control of a strong promoter recognized by T7 phage RNA polymerase.

The fusion (His) × 10_IntMUT_Ssm6A contains no linker between the last amino acid residue of intein (Asn) and the first residue of mature Ssm6A (Ala).

The cultivation of the strain containing this design and analysis of the expressed fusion is carried out according to the method described in Example 4.

In FIG. Figure 8 shows the accumulation in the insoluble fraction of exclusively unprocessed His × 10_IntMUT_Ssm6A protein with a molecular weight of about 25 kD.

Further operations to clean inclusion bodies containing fusion (His) × 10_intMUT_Ssm6A, as well as its processing, are carried out according to the procedure described in Example 4 for protein (His) × 10_IntMUT_SUMO.

From the results shown in FIG. 8, it can be seen that the obtained protein (His) × 10_IntMUT_Ssm6A is able to be efficiently processed in vitro. Moreover, the fraction of processed protein (His) × 10_IntMUT_Ssm6A determined by protein electrophoresis is about 35%, which is lower than the fraction of processed protein (His) × 10_IntMUT_SUMO (Fig. 3) and illustrates the dependence of the processing efficiency of the protein precursor on the properties of the target protein.

Example 8. Expression and processing in vitro protein (His) × 10_IntMUT_CVIE

The effectiveness of the claimed intein for the insoluble expression of the precursor protein is confirmed by the synthesis of another model target protein, the recombinant neurotoxin CVIE from the snail venus Conus catus, the amino acid sequence and structure of which are disclosed in [26].

For this purpose, a recombinant plasmid pET28b_ (His) × 10_IntMUT_CVIE containing a complex DNA fragment encoding a fusion protein (His) × 10_IntMUT_CVIE is constructed.

The complex DNA fragment is obtained in two stages.

At the first stage, two overlapping DNA fragments are constructed:

• Fragment 1 is obtained as described in example 7;

• Fragment 2, comprising a DNA sequence containing a unique XhoI site at the 3-terminal portion and encoding a CVIE protein, is synthesized as a double-stranded DNA sequence during PCR using the following set of overlapping oligonucleotides:

The amplified DNA fragments were purified on an agarose gel using a Qiagen whale (Qiagen, cat. No. 28706) and used in a second step to prepare a complex DNA fragment.

At this stage, a mixture of purified overlapping fragments 1 and 2 is introduced into the PCR reaction mixture. N (his) and N (ter2) are used as amplification primers. As a result of PCR, fragments 1 and 2 are ligated and a complex amplified DNA fragment of 620 bp is obtained, encoding fusion (His) × 10_IntMUT_CVIE.

The resulting DNA fragment was purified on an agarose gel, digested with restriction enzymes NcoI and XhoI and cloned in the expression vector pET28b (Novagen) at the restriction sites NcoI and XhoI.

Cloning yields the plasmid pET28b_ (His) × 10_IntMUT_CVIE, which contains the fusion gene (His) × 10_IntMUT_CVIE under the control of a strong promoter recognized by T7 phage RNA polymerase.

The fusion (His) × 10_IntMUT_Ssm6A contains no linker between the last amino acid residue of intein (Asn) and the first residue of mature CVIE (Cys).

The cultivation of the strain containing this design and analysis of the expressed protein is carried out according to the method described in Example 4.

In FIG. Figure 9 shows the accumulation in the insoluble fraction of the exclusively unprocessed His × 10_IntMUT_Ssm6A protein with a molecular weight of about 24 kD.

Further operations to clean inclusion bodies containing fusion (His) × 10_intMUT_CVIE, as well as its processing, are carried out according to the procedure described in Example 4 for protein (His) × 10_IntMUT_SUMO.

From the results shown in FIG. 9, it is seen that the obtained protein (His) × 10_IntMUT_CVIE is able to be efficiently processed in vitro. Moreover, the fraction of processed protein (His) × 10_IntMUT_CVIE determined by protein electrophoresis is about 60%, which exceeds the fraction of processed protein (His) × 10_IntMUT_SUMO (Fig. 3) and illustrates the dependence of the processing efficiency of the protein precursor on the properties of the target protein.

Thus, for the implementation of the insoluble expression of the target protein precursor, which is a fusion of a self-cleaving protein module with a target protein, the claimed temperature-sensitive mutant intein (SEQ ID NO 1) is proposed as a self-cleaving protein module.

It was shown that the degree of fusion processing of the claimed mutant intein with the target protein varies depending on the selected target protein. For the model target protein SUMO, this value is more than 50%, and for the neurotoxin CVIE - about 60%.

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[26] Berecki G, Motin L, Haythornthwaite A, Vink S, Bansal P, Drinkwater R et al. (2010). Analgesic ω-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels. Mol Pharmacol, 77: 139-148.

Claims (1)

Temperature-sensitive mutant PRP8 mini-intein from Penicillium chrysogenum, for insoluble expression of the target protein precursor in Escherichia coli cells, comprising replacing the amino acid residue of leucine at position 93 with a proline residue, replacing the cysteine residue at position 1 with an alanine residue, and optionally containing replacing the cysteine residue at position 11 per tyrosine residue in the original PRP8 sequence of mini-intein from Penicillium chrysogenum, consisting of 157 amino acid residues.

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