Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid–protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K+ channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~ 80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of 15N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.

The gene for the “weak” toxin of Naja kaouthia venom was expressed in Escherichia coli. “Weak” toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of “weak” toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia “weak” toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain “weak” toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of 125I-labeled α-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (α12β1γδ) showed the presence of biological activity of the recombinant “weak” toxin close to the activity of the natural toxin (IC50 = 4.3 ± 0.3 and 3.0 ± 0.5 µM, respectively). The interaction of the recombinant toxin with α7 type human neuronal acetylcholine receptor transfected in the GH4C1 cell line also showed the presence of activity close to that of the natural toxin (IC50 31 ± 5.0 and 14.8 ± 1.3 µM, respectively). The developed bacterial system for production of N. kaouthia venom “weak” toxin was used to obtain 15N-labeled analog of the neurotoxin.

Экспрессирован ген «слабого» токсина из яда кобры Naja kaouthia. «Слабый» токсин является специфичным ингибитором никотинового ацетилхолинового рецептора, однако механизмы взаимодействия подобных нейротоксинов с рецептором на данный момент остаются неизвестными. Для бактериальной продукции «слабого» токсина из яда кобры N. kaouthia опробованы системы, разработанные ранее для нейроток-сина II из яда кобры N. oxiana. Созданы конструкции для цитоплазматической продукции «слабого» токсина из яда кобры N. kaouthia в виде слитной полипептидной цепи с тиоредоксином и для секреции с лидер-ным пептидом STII. Однако получить «слабый» токсин в миллиграммовых количествах удалось только в составе цитоплазматических телец включения. Опробованы различные подходы для ренатурации токсина и проведен поиск условий оптимизации выхода целевого белка в процессе ренатурации. Полученный белок охарактеризован методами масс-спектроскопии, КД- и ЯМР-спектроскопии. Эксперименты по конкурентному ингибированию связывания [1251]б-бунгаротоксина с мембранами электрического органа ската Torpedo californica, содержащими никотиновый ацетилхолиновый рецептор мышечного типа (б12Я1гд), показали наличие биологической активности рекомбинантного «слабого» токсина, близкой к активности природного токсина (IC50 4,3 ± 0,3 и 3,0 ± 0,5 мкМ соответственно). Взаимодействие рекомбинантного токсина с нейрональным никотиновым ацетилхолиновым рецептором человека а7 типа, трансфецированным в клеточной линии GH4C1, также показало наличие активности, близкой к таковой природного токсина (IC50 31 ± 5 и 14,8 ± 1,3 мкМ соответственно). Разработанная бактериальная система продукции «слабого» токсина из яда кобры N. kaouthia позволила получить 15N-меченный аналог «слабого» токсина.

Проведена ренатурация биотехнологического предшественника инсулина человека из раствора телец включения без предварительного сульфирования или восстановления остатков цистеина. Тельца включения растворили в 8 М мочевине с добавлением 10 мМ 2-меркаптоэтанола. Из раствора удалили гидрофобные клеточные компоненты пропусканием через нейтральный слабогидрофобный сорбент, раствор разбавили в 5 раз и ренатурировали с добавлением 0.3 мМ цистина для инициации реакции тиол-дисульфидного обмена. Присутствие нуклеиновых кислот и примесных клеточных белков не оказывает влияния на ренатурацию. Полученный ренатурированный предшественник инсулина человека очищен металлхелатной аффинной хроматографией и превращен в инсулин двухступенчатым ферментативным расщеплением.

The folding of biotechnological precursor of the human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing its Cys residues. The inclusion bodies were dissolved in 8 M urea with the addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.

The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins—biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency. However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography. In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents. A negative influence of nucleic acid and heavy metal ions on folding has been found. S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding. Folded fusion proteins are transformed into insulin by enzymatic cleavage.

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein. The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation. Folding kinetics of IFN-gamma was studied by reversed-phase HPLC. IFN-gamma and mutant proteins were characterized by their thermal stability and biological activity. Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19 degrees C and four times enhancement of biological activity compared with intact IFN-gamma molecule.

Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized. Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions. An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy. It was thereby found that at pH 8.5, which is the optimal value for renaturation, the protein has the secondary structure most close to the native one. The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 × 10 7 U/mg.

Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide. Six residues of Cys in proinsulin allow the chemical modification of the protein as a (Cys-S-SO(-)(3))(6) derivative (S-sulfonate), which increases its polyelectrolytic properties and improves the efficiency of its isolation. Various methods of oxidative sulfitolysis were compared with catalysis by sodium tetrathionate or cystine and Cu2+ or Ni2+ ions. An optimum scheme for the isolation and purification of S-sulfonated fusion proteins was developed by the combination of metal-chelating affinity and ion-exchange chromatography. Highly purified (95%) S-sulfonated fusion protein was recovered which was 85% of the fusion protein contained in the biomass of E. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S-sulfonate has proinsulin-like secondary structure. This structure causes highly efficient fusion protein folding.

Two schemes for efficient and productive isolation for mutant human recombinant tumor necrosis factor-alpha (TNF-alpha R32H) from Escherichia coli cells were developed. The methods include membrane filtration, ion-exchange chromatography and gel filtration, and centrifugation with subsequent free-flow electrophoresis as an alternative procedure. The target product was obtained as active trimer with total yield more than 50% and greater than 98% purity according to PAGE, size-exclusion chromatography, HPLC, and HPCE.

An efficient method for the isolation, purification, and renaturation of human recombinant gamma-interferon from biomass of transformed E. coli cells was developed. It involves the extraction of the protein from the inclusion bodies, preliminary purification of the protein, and three stages of ion-exchange chromatography with an intermediate renaturation between the second and the third stages. A highly active (2 x 10(7) U/mg) recombinant protein of up to 99% purity (according to SDS-PAGE and HPLC) was obtained with a 30% overall yield.

An efficient method for the isolation, purification, and renaturation of human recombinant gamma-interferon from biomass of transformed E. coli cells was developed. It involves the extraction of the protein from the inclusion bodies, preliminary purification of the protein, and three stages of ion-exchange chromatography with an intermediate renaturation between the second and the third stages. A highly active (2 x 10(7) U/mg) recombinant protein of up to 99% purity (according to SDS-PAGE and HPLC) was obtained with a 30% overall yield.

An efficient and productive isolation method for human recombinant tumor necrosis factor alpha from Escherichia coli cells was developed. The method includes a membrane filtration step, two steps of ion-exchange chromatography, and gel filtration on a Sephadex G-25 column. The target product was obtained with approximately 50% total yield and greater than 95% purity according to PAGE and HPLC.

An efficient and productive isolation method for human recombinant tumor necrosis factor a from Escherichia coli cells was developed. The method includes a membrane filtration step, two steps of ion-exchange chromatography, and gel filtration on a Sephadex G-25 column. The target product was obtained with approximately 50% total yield and greater than 95% purity according to PAGE and HPLC.