RU2740715C2 - Modified groel protein of microorganism thermus thermophilus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to thermally stable protein GroEL of Thermus thermophilus microorganism. Disclosed is a thermally stable GroEL protein of a microorganism Thermus thermophilus, used as carrier protein of target peptides in fused protein structures for expression in host microorganism cells. Coding DNA sequence of said protein contains a polylinker GGATCCAAGCTTGAATTC between codons corresponding to positions Ser199 and Tyr201, wherein the polylinker sequence contains restriction sites HindIII, BamHI, EcoRI, and corresponds to the sequence Gly-Ser-Lys-Leu-Glu-Phe.

EFFECT: invention provides higher stability, thermal stability and expression level of said GroEL protein in a host cell.

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Description

The invention relates to the field of biochemistry, namely, to obtain a new, modified form of the thermostable protein GroEL of the microorganism T. thermophilus. It can be used in the pharmaceutical industry as a carrier protein for the biosynthesis of recombinant peptides, the synthesis of which is not profitable by existing technologies, in research laboratories for the production of various peptides in order to search for those with the necessary properties.

The GroEL family of chaperone proteins looks attractive as a carrier protein for a fusion protein system [Alexey N. Fedorov, and Maria S. Yurkova. Molecular Chaperone GroEL - toward a Nano Toolkit in Protein Engineering, Production and Pharmacy // Nano World J. 2018. T. 4. No. 1. C. 8-15]. This chaperone is a large particle composed of two heptamers. The cavity formed by the protomer contains the protein substrate. GroEL is a well-studied chaperone, its structure and functioning are described in detail [Horwich A.L., Farr G.W., Fenton W.A. GroEL-GroES-mediated protein folding // Chem. Rev. 2006. T. 106. No. 5. C. 1917-1930; Horwich A.L. et al. Two families of chaperonin: physiology and mechanism // Annu Rev Cell Dev Biol. 2007. T. 23. C. 115-145]. Typically, one protein substrate molecule binds to one GroEL protomer; as for the whole particle, usually negative cooperativity is observed between the two protomers when binding the substrate protein. The total cavity formed inside the heptamer is sufficient to accommodate a sufficiently large substrate protein [Thirumalai D., Lorimer G.H. Chaperonin-mediated protein folding // Annu. Rev. Biophys. Biomol. Struct. 2001. T. 30. S. 245-269; Saibil N. Chaperone machines for protein folding, unfolding and disaggregation // Nat. Rev. Mol. Cell Biol. 2013. T. 14. No. 10. C. 630-642].

The attachment of additional amino acid sequences to the GroEL sequence affects the stability of the chaperone. Both its N-terminus and C-terminus are deeply embedded in the protein globule, so that the attachment of any additional sequences to either of the ends can affect the tertiary and quaternary structures. Several research results indicate that placement of the 6His-tag destabilizes the original structure of the GroEL particles and that the His-tagged GroEL is purified with low efficiency and results in only the monomeric form. Studying the three-dimensional structure of GroEL suggests that attaching any additional sequences to its ends should destabilize its structure. The main goal of this study was to achieve the ability of GroEL to be a carrier protein for the attachment of target peptides, while maintaining its tertiary and quaternary structures. The only study that previously addressed this issue was based on the analysis of random insertions of transposons into the GroEL genome. It was found that transposon insertions destabilized the GroEL particle, they all led to destabilization of the heptamer rings, many mutants were insoluble [Amatore D., Baneyx F. Insertion mutagenesis of Escherichiacoli GroEL // Biochem. Biophys. Res. Commun. 2003. T. 302. No. 2. S. 246-252]. The natural limitation of this approach and, as a consequence, the limitation of its conclusions follows from the analysis of a limited number of transposon variants that were not preselected for optimal placement of the insert taking into account the tertiary structure. It is known that random transposon insertions usually inactivate proteins and affect their native structure.

показывает, что каждая субъединица имеет три домена. В большинстве организмов GroEL имеет сложную четвертичную структуру, состоящую из двух колец по семь мономеров в каждом, и стабилизированную кольцом из семи мономеров ко-фактора GroES (фиг. 1)Crystal structure analysis of GroEL monomer with 2.8 resolution

shows that each subunit has three domains. In most organisms, GroEL has a complex quaternary structure consisting of two rings of seven monomers each, and stabilized by a ring of seven monomers of the GroES co-factor (Fig. 1)

The technical result of the invention is to increase the stability, thermal stability and expression level of the modified GroEL protein in the host cell, reduce the sensitivity of amino acid sequences to proteolysis, as well as the mandatory content in the coding part of the polylinker for the possibility of integrating the target peptide sequence.

The technical result of the invention is achieved by creating a construct of the GroEL chaperone version of the microorganism T. thermophilus, by changing the GroEL DNA coding sequence - a polylinker with the GGATCCAAGCTTGAATTC sequence was integrated between the codons at positions Ser199 and Tyr201. This sequence contains the BamHI, HindIII and EcoRI restriction sites and corresponds to the transcription of Gly-Ser-Lys-Leu-Glu-Phe. Analysis of the tertiary structure of the GroEL monomer shows that the amino acid residues Ser199 and Tyr201 are located on the subtrate-binding surface of the apical domain of GroEL, but are not part of the rigid structures that form the substrate-binding surface. In the stable GroELS particle, the amino acid residues Ser199 and Tyr201 are located on the surface facing the interior of the particle cavity (Fig. 2), which makes it possible to subsequently use the modified protein as a carrier protein in fusion constructs with the target peptide. The GroELS particle has a high molecular weight (more than 850 kDa), which technologically simplifies the isolation of the construct from the cell lysate by gel filtration and ultrafiltration. The use of a chaperone of a thermophilic microorganism, in addition, allows the first stage of purification by thermal denaturation of endogenous proteins of the mesophilic bacterium.

The modified protein after insertion of the polylinker should not change the native conformation, but should remain stable and well soluble.

The essential features that characterize the invention, in contrast to the original version of the protein, is the use of a modified thermostable protein GroEL of the microorganism T. thermophilus with the presence of a polylinker for the possibility of using it to create constructs with a target peptide, highly expressive, not subject to proteolysis, with neutralization of any peptide cytotoxicity.

Brief description of figures and drawings:

FIG. 1 The structure of the GroELS complex.

FIG. 2 Protomer GroEL. The sites of polylinker injection on the substrate-binding surface of the apical domain were identified.

FIG. 3 Expression of GroEL with integrated sequence of polylinker 1 - before IPTG induction 2 - after IPTG induction.

FIG. 4 Electrophoresis of the protein profile of the modified GroEL with an integrated polylinker. 1 - cell lysate before heating, 2 - supernatant after heating, 3 - sediment after heating, MW - protein molecular weight marker.

FIG. 5 Expression of GroEL with an integrated polyphemusine sequence at polylinker position 1 - before IPTG induction 2 - after IPTG induction.

FIG. 6 Electrophoresis of the protein profile of modified GroEL with integrated polyphemusine. 1 - cell lysate before heating, 2 - supernatant after heating, 3 - sediment after heating, MW - protein molecular weight marker.

The proposed method is illustrated by the following materials:

A polylinker was introduced into the GroEL gene. A small polylinker containing BamHI, HindIII and EcoRI was introduced for cloning into the gene between the triplets encoding the amino acids Ser199 and Tyr201. In a first step, two segments of the desired gene construct were separately amplified using T. Thermophilus DNA as template (RefSeq assembly: GCF_000091545.1; GenBank: AP008226.1). The first fragment was amplified using primers SeqID # 1-2, the second using primers SeqID # 3-4. The reactions were carried out under the following conditions: Pfu buffer 1x (20 mM Tris-HCl pH = 8.8; 10 mM (NH ₄ ) SO ₄ , 10 mM KCl, 0.1% Triton X-100 v / v, 0.1 mg / ml BSA), 1.2 u Pfu-polymerase, 10 pmol each primers, according to the following PCR scheme: 94 ° C 3 minutes, 40 cycles (94 ° C 40 sec, 60 ° C 40 sec, 72 ° C 2 minutes), 72 ° C 3 minutes.

Fragments were purified by agarose gel electrophoresis in 1% agarose gel in 1x TBE buffer, the fragment was purified using a kit for DNA purification from QIAEX II Gel Extraction Kit

Combination amplification using the purified fragments obtained in the previous step, in the form of templates and two primers SeqID # 1 and SeqID # 4, produced a nucleotide gene sequence encoding GroEL with a polylinker. The reaction was carried out under the following conditions: Pfu buffer 1x (20 mM Tris-HCl pH = 8.8; 10 mM (NH ₄ ) SO ₄ , 10 mM KCl, 0.1% Triton X-100 v / v, 0.1 mg / ml BSA), 1.2 u Pfu-polymerase, 10 pmol each primers, according to the following PCR scheme: 94 ° C 3 minutes, 40 cycles (94 ° C 40 sec, 60 ° C 40 sec, 72 ° C 2 minutes), 72 ° C 3 minutes.

The fragment was purified by agarose gel electrophoresis in 1% agarose gel in 1x TBE buffer using a kit for DNA purification from gel "QIAEX II Gel Extraction Kit"

Then the resulting fragment was digested with restriction enzymes NdeI and BglII under the following conditions: 1x buffer O (Orange) (50 mM Tris-HCl pH = 7.5, 10 mM MgCl ₂ , 100 mM NaCl, 0.1 mg / ml BSA), 5 units each. each enzyme BglII and NdeI, 50 ng DNA and incubated at 37 ° C for 16 hours.

GroES was amplified using primers SeqID # 5-6 containing restriction sites for NcoI and NotI, respectively, and the T. thermophilus genome as a template under the following conditions: Pfu buffer 1x (20 mM Tris-HCl pH = 8.8; 10 mM (NH ₄ ) SO ₄ , 10 mM KCl, 0.1% Triton X-100 v / v, 0.1 mg / ml BSA), 1.2u Pfu-polymerase, 10 pmol of each primer, according to the following PCR scheme: 94 ° C 3 minutes, 40 cycles ( 94 ° C 40 sec, 60 ° C 40 sec, 72 ° C 2 minutes), 72 ° C 3 minutes. The resulting PCR fragment was digested with NcoI and NotI enzymes under the following conditions: Tango buffer 2x (66 mM Tris-acetate pH = 7.9, 20 mM magnesium acetate, 132 mM potassium acetate, 0.2 mg / ml BSA), 5 units. each enzyme NcoI and NotI; incubated at 37 ° C for 16 hours. Then the bicistronic vector pET Duet was cloned into the first expression polylinker and the expression vector pGroES was obtained. Ligation was performed under the following conditions: Ligation buffer 1x (40 mM Tris-HCl pH = 7.9, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM ATP), 20 ng plasmid, 1: 5 molar vector-to-insert ratio, 2 units a ... Weiss ligases. Ligation was performed at 22 ° C for 1 hour.

Then the GroEL fragment with the integrated target polylinker was cloned into the polylinker of the pET Duet bicistronic expression vector together with the gene encoding T. thermophilus GroES. Ligation was performed under the following conditions: Ligation buffer 1x (40 mM Tris-HCl pH = 7.9, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM ATP), 20 ng plasmid, 1: 5 molar vector-to-insert ratio, 2 units a ... Weiss ligases. The ligation was performed at 22 ° C for 1 hour, which gives the expression vector ploop / ES.

Chemically competent cells of the E. coli strain pLysE in a volume of 50 μl were thawed on ice, then 5 μl of ligase mixture was added and incubated for 30 minutes on ice. Then, thermal shock was performed at 42 ° C for 30 seconds, followed by incubation on ice for 5 minutes. Next, 1 ml of SOC medium preheated to 37 ° C was added to the transformants and incubated for 1 hour with constant rocking in a rocking thermostat. Next, 20 μl of the incubated mixture was plated onto plates with LB solid culture medium containing 100 μg / ml ampicillin. Colonies were grown overnight at 37 ° C. Then, analytical expression was carried out for individual clones with the inducer of 0.4 mM IPTG (Fig. 3).

Expression of genes in the expression strain of E. coli pLysE was carried out in LB medium, at 37 ° C for 3 hours; the selective agent is ampicillin 100 μg / ml.

To purify the target protein, lysis was carried out using a French press in PBS buffer (0.137 M NaCl, 0.0027 M KCl, 0.01 M Na ₂ HPO ₄ , 0.0018 M KH2PO4) at 4 ° C. It was then diluted 5 times with PBS buffer, followed by heating the cell lysate at 62 ° С for 5 min in a water bath, and then centrifuged at 13000g for 15 min. (fig. 4)

To confirm the functionality of the fusion construct, the polypeptide "Polyphemusin", known for its bactericidal activity, was inserted into the restriction zones of the integrated polylinker. The sequence of polyphemusin SeqID # 7 was optimized for use in E. coli. This polyphemusin variant was hybridized from two SeqID # 8-9 oligonucleotides. The oligonucleotides were designed so that the resulting polyphemusine had ready-made sticky ends for cloning into the BamHI / EcoRI sites of the polylinker introduced into the GroEL gene. The bicycron vector construct was digested with restriction enzymes BamHI and EcoRI, and polyphemusine was cloned into it. All results from cloning procedures were confirmed by sequencing.

Gene expression in the expression strain of E. coli pLysE was carried out in LB medium, at 37 ° C for 3 hours; the selective agent is ampicillin 200 μg / ml. (fig. 5)

The polypeptide was purified by heating the cell lysate at 62 ° C for 5 min in a water bath, followed by centrifugation at 13000 g for 15 min. (fig. 6)

Example 1. Obtaining a modified GroEL protein

Stage 1.

Separately amplify using T. Thermophilus DNA as template (RefSeq assembly: GCF_000091545.1; GenBank: AP008226.1) the first fragment using SeqID # 1-2 primers, the second using SeqID # 3-4 primers. Carry out the reaction under the following conditions: Pfu buffer 1x (20 mM Tris-HCl pH = 8.8; 10 mM (NH ₄ ) SO ₄ , 10 mM KCl, 0.1% Triton X-100 v / v, 0.1 mg / ml BSA), 1.2 u Pfu-polymerase, 10 pmol each primers, according to the following PCR scheme: 94 ° C 3 minutes, 40 cycles (94 ° C 40 sec, 60 ° C 40 sec, 72 ° C 2 minutes), 72 ° C 3 minutes.

Purify the fragments by agarose gel electrophoresis in 1% agarose gel in 1x TBE buffer using a kit for DNA purification from gel "QIAEX II Gel Extraction Kit"

Carry out combination amplification using the purified fragments obtained at the previous stage in the form of templates and two primers SeqID # 1 and SeqID # 4 under the following conditions: Pfu buffer 1x (20 mM Tris-HCl pH = 8.8; 10 mM (NH ₄ ) SO ₄ , 10 mM KCl, 0.1% Triton X-100 v / v, 0.1 mg / ml BSA), 1.2u Pfu-polymerase, 10 pmol each primers, according to the following PCR scheme: 94 ° C 3 minutes, 40 cycles (94 ° 40 sec, 60 ° C 40 sec, 72 ° C 2 minutes), 72 ° C 3 minutes.

The fragment is purified by agarose gel electrophoresis in 1% agarose gel in 1x TBE buffer using a kit for DNA purification from gel "QIAEX II Gel Extraction Kit"

Stage 2.

The obtained fragment is digested with restriction enzymes NdeI and BglII under the following conditions: 1x buffer O (Orange) (50 mM Tris-HCl pH = 7.5, 10 mM MgCl ₂ , 100 mM NaCl, 0.1 mg / ml BSA), 5 units each. each enzyme BglII and NdeI, 50 ng DNA and incubate at 37 ° C for 16 hours.

Stage 3.

Amplify GroES using primers SeqID # 5-6 containing restriction sites for NcoI and NotI, respectively, and the T. thermophilus genome as a template under the following conditions: Pfu buffer 1x (20 mM Tris-HCl pH = 8.8; 10 mM (NH ₄ ) SO ₄ , 10 mM KCl, 0.1% Triton X-100 v / v, 0.1 mg / ml BSA), 1.2u Pfu-polymerase, 10 pmol of each primer, according to the following PCR scheme: 94 ° C 3 minutes, 40 cycles ( 94 ° C 40 sec, 60 ° C 40 sec, 72 ° C 2 minutes), 72 ° C 3 minutes. The obtained PCR fragment is digested with NcoI and NotI enzymes under the following conditions: Tango buffer 2x (66 mM Tris-acetate pH = 7.9, 20 mM magnesium acetate, 132 mM potassium acetate, 0.2 mg / ml BSA), 5 units of a. each enzyme NcoI and NotI; incubated at 37 ° C for 16 hours. Then clone the bicistronic vector pET Duet into the first expression polylinker and obtain the pGroES expression vector. Ligation is performed under the following conditions: Ligation buffer 1x (40 mM Tris-HCl pH = 7.9, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM ATP), 20 ng plasmid, 1: 5 molar vector-insert ratio, 2 units a ... Weiss ligases. Carry out ligation at 22 ° C for 1 hour.

Stage 4.

Clone the GroEL fragment with the integrated target polylinker obtained in step 2 into the polylinker of the bicistronic expression vector pET Duet together with the gene encoding T. thermophilus GroES. Ligation is performed under the following conditions: Ligation buffer 1x (40 mM Tris-HCl pH = 7.9, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM ATP), 20 ng plasmid, 1: 5 molar vector-insert ratio, 2 units a ... Weiss ligases. Carry out ligation at 22 ° C for 1 hour.

Stage 5.

Thaw chemically competent cells of the E. coli strain pLysE in a volume of 50 μl on ice, then add 5 μl of the ligase mixture obtained in step 4 and incubate for 30 minutes on ice. Then conduct thermal shock at 42 ° C for 30 seconds, followed by incubation on ice for 5 minutes. Next, add 1 ml of SOC medium preheated to 37 ° C to the transformants and incubate for 1 hour with constant rocking in a rocking thermostat. Next, dispense 20 μl of the incubated mixture onto plates with solid LB culture medium containing 100 μg / ml ampicillin. Grow colonies overnight at 37 ° C. Then carry out analytical expression for individual clones, with the inductor 0.4 mM IPTG.

The developed variant of GroEL is a protein consisting of 548 amino acid residues with a molecular weight of 58370 Daltons.

Claims (1)

Thermostable protein GroEL of the microorganism Thermus thermophilus, used as a carrier protein of the target peptides in fusion protein constructs for expression in the cells of the host microorganism, the coding DNA sequence of which contains the polylinker GGATCCAAGCTTGAATTC between the codons corresponding to positions Ser199 and Tyr201, while the sites of the polylinker HindIII , BamHI, EcoRI, and corresponds to the sequence Gly-Ser-Lys-Leu-Glu-Phe.

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