Department of biotechnology

All publications (show selected)

Anatoliy Miroshnikov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 53, office. 6601
  • Phone: +7(495)995-55-57#2005
  • E-mail: aiv@ibch.ru

Preparation of active pharmaceutical ingridient based on Nα-acetyl C-amide terminal form of HLDF-6 peptide using optimized synthetic method

Laboratory of hormonal regulation proteins,  Group of Peptide Chemistry

Standard solid phase methods could not be applied for the preparation of the API samples due to the fact that current SPPS methodologies are still imperfect. Therfore, fragment condensation in solution method was developed, optimized and used for the preparation of the amide form of HLDF-6 peptide. That material could be purified and acetylated under the conditions where peptide salts of weak acids are acylated with variuos weak electrophilic derivatives of acetic acid including active esters and azolides. Appliaction of the acetylation reaction conditions described provides API samples in high yield and purity

Publications

  1. Zolotarev YuA, Dadayan AK, Kozik VS, Shram SI, Nagaev IYu, Azev VN, Bogachouk AP, Lipkin VM, Myasoedov NF (2019). Proteolytic Hydrolysis of the Antitumor Peptide HLDF-6-AA in Blood Plasma. Russ. J. Bioorganic Chem. 45 (5), 347–352

Сalcium-induced activation of the bacteriophage T5 peptidoglycan hydrolase promoting host cell lysis

Group of Molecular Biotechnology

The calcium activation of the bacteriophage T5 endolysin was studied. A non-canonical EF-like Cа2+-binding loop was detected in the enzyme structure using site-directed mutagenesis. Taking into account the role of calcium ion we proposed the new model for the regulation of phage-mediated lysis of the host bacteria cell wall. The results suggest that Ca2+ binding to EndoT5 molecule could be essential for stabilization of the long mobile loop in catalytically active "open" conformation. The study reveals valuable insight for the role of calcium in the regulation of phage-induced bacterial lysis.

Publications

  1. Kovalenko AO, Chernyshov SV, Kutyshenko VP, Molochkov NV, Prokhorov DA, Odinokova IV, Mikoulinskaia GV (2019). Investigation of the calcium-induced activation of the bacteriophage T5 peptidoglycan hydrolase promoting host cell lysis. Metallomics 11 (4), 799–809

A convenient approach to the biosynthesis of C2,C6-disubstituted purine nucleosides using E. coli purine nucleoside phosphorylase and arsenolysis

Laboratory of biosynthesis of physiologically active compounds

A method of synthesis of new of 3'-deoxyribonucleosides from corresponding ribosides by enzymatic transglycosylation is suggested. It is shown that the use of orthoarsenic acid salts allows to shift the equilibrium of the enzymatic reaction towards the formation of target nucleosides with an efficiency of up to 90% conversion. A scheme of synthesis of 3'-desoxyribose 6-methoxypurine is shown on the figure. In scientific practice arsenates are used exclusively for the degradation of ribo- and deoxyribonucleosides. Only in our laboratory just the second technology in which arsenate is used for highly effective synthesis of new nucleosides is offered.

Manuscript "A convenient approach to the biosynthesis of C2,C6-disubstituted purine nucleosides using E. coli purine nucleoside phosphorylase and arsenolysis» will be published in  Book “Applied Biocatalysis: The Chemist's Enzyme Toolkit”, Fourth Edition, 2020, Publisher Wiley-VCH.

Effect of C-terminal His-tag and purification routine on the activity and structure of the bacteriophage T5 endolysin

Group of Molecular Biotechnology

The effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, bacteriophage T5 endolysin, whose zinc binding site and catalytic aspartate are located near the C-terminus, were studied. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, but reduced stability and half-life. However, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying IMAC. The data obtained will be useful to a wide range of researchers working with recombinant proteins.

Two novel thermally resistant phage-specific peptidases

Group of Molecular Biotechnology

Two novel enzymes, peptidoglycan hydrolases of bacteriophages RB43 and RB49 (Myoviridae), were obtained and biochemically characterized. These are zinc-containing l-alanyl- d-glutamate peptidases belonging to the M15 family. Both enzymes are resistant to high temperatures and effectively hydrolyze peptidoglycan of Gram-negative bacteria, which makes them promising for use in biotechnology and biomedicine.

Publications

  1. Mikoulinskaia GV, Chernyshov SV, Shavrina MS, Molochkov NV, Lysanskaya VY, Zimin AA (2018). Two novel thermally resistant endolysins encoded by pseudo T-even bacteriophages RB43 and RB49. J Gen Virol 99 (3), 402–415

Development of efficient solid-phase synthesis methods for the preparation of peptides that possess immunosuppresive activity

Laboratory of Biological Testing,  Laboratory of pharmacokinetics,  Group of Peptide Chemistry

The relative efficacies of several synthesis methods have been investigated aiming to prepare target peptides that are quite potent in a test for suppression of the experimental autoimmune encephalomyelitis. Toward this goal chemical yields of the target peptides as well as side product distributions in the samples of the peptides obtained using various methods have been evaluated and quantitatively characterized. A variation of Fmoc/tBu methodology have been found to be the most efficient providing targets peptides in the highest chemical yield since it allowed to use a broader range of  activated amino acid derivatives. Side products of the amino acid doubling were detected among the side products of chemical synthesis. Some of the amino acid doubling side products made the HPLC purification step to be complicated. Nevertheless the synthetic methods developed allowed the preparation of the target peptides in sufficient quantities.

A new approach for the synthesis of biologically important nucleotides using a thermophilic multi-enzymatic cascade

Laboratory of Biopharmaceutical Technologies

A new strategy of cascade synthesis of modified nucleotides was implemented using enzymes of thermophilic microorganisms (ribokinase RK from Thermus species 2.9, phosphoribosylpyrophosphate synthetase (PRPPS) and adenine phosphoribosyltransferase (APRT)/hypoxanthine phosphoribosyltransferase (HPRT) from Thermus thermophilus HB27) carry out successive transformations of ribose and purine heterocyclic bases into corresponding nucleotides. The comprehensive studies of determination of the kinetic parameters of enzyme- catalyzed reaction and optimization of the conditions for the cascade synthesis of modified nucleotides were performed. The potential nucleotide 5'-monophosphates with antiviral and anti- cancer activities were synthesized using multi-enzymatic cascade. Crystallization of thermophilic apo-enzymes and enzymes-ligands complex and subsequent X-ray structural analysis were carried out. Structures of the APRT and complex PRPPS/ADP/SO 4 2- were solved and deposited into RCSB Protein Data Bank with PDB entries 6fsp (at 2.5 Å resolution) and 5t3o (at 2.2 Å resolution), respectively. Three-dimensional structure of the ribokinase was solved at 1.9 Å resolution.

Development of methods for obtaining thymosin beta 4 analogs as conjugates with extended half-life in vivo

Laboratory of Biopharmaceutical Technologies

A method for the production of monocojugates of recombinant human thymosin β4 (Тβ4) with capronic acid, polysialic acid 14 kDa and polyethylene glycol 10 kDa has been developed. As a result of multifactorial experiments, the components of the reaction mixtures were identified, and conditions of those reactions were optimized. For each analog, we developed a single-stage RP HPLC purification procedure that enabled to attain the purity of no less than 98%. The peptide mapping results backed up by chromatography-mass spectrometry and electrophoresis demonstrated that the produced Тβ4 analogs are monoconjugates in which the initial peptide is modified at the N-terminal serine residue. The produced analogs feature a higher resistance to degradation in the blood plasma as compared to non-modified Tβ4, and they can be regarded as promising candidates for further biological testing.

Multiple sclerosis is a chronic autoimmune disease with neurological pathology. The dominant role of immunological processes in the development of the disease dictates the need for medications that specifically minimize the activity of immune processes. The peptides A8AMS and mA8AMS, homologous to the fragment of the human IgG VH domain, have been shown to act in vitro and in vivo, effectively reducing the symptoms of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. The results provide new opportunities for the development of peptide drugs for the treatment of multiple sclerosis.