Kolomiiets L. Creation and characterization of a new nanocomposite complexЕМАРІІ with dextran 70

Українська версія

Thesis for the degree of Candidate of Sciences (CSc)

State registration number

0420U102370

Applicant for

Specialization

  • 03.00.20 - Біотехнологія

15-12-2020

Specialized Academic Board

Д 26.237.01

Institute of Molecular Biology and Genetics of NAS of Ukraine

Essay

Endothelial monocyte activating polypeptide II (EMAP II) is a cytokine capable of exhibiting anti-angiogenic action, inducing expression of TNF and interleukin-8, causing apoptosis of endothelial cells. EMAP II ability to inhibit neoangiogenesis and stimulate cancer cell apoptosis has become the basis for investigating whether it can be used as an anticancer drug. The antitumor effect of EMAP II has been experimentally demonstrated on different types of tumors. Proteins and protein preparations are widely used in medical practice in the treatment of various diseases. The main problem with the use of proteins as drugs is their instability, high ability to aggregate and low degree of solubility.The most effective way of combating the problem of aggregation is to use variety of ligands that prevent aggregation. Dextran belongs to the family of natural polysaccharides that are widely used in clinical and preclinical studies with a well-characterized safety profile. Dextran is also used in nanomedicine, which uses submicron particles for therapeutic and diagnostic purposes. Dextran is also used to protect and stabilize the unique structure of the proteins (e.g., albumin, streptokinase, asparagine, insulin, hemoglobin). The bacterial expression of EMAP II cytokine and preparation of recombinant protein in preparative amounts were optimized. It was shown that the optimal conditions for cytokine expression are the cultivation of bacterial culture of E. coli BL21 (DE3) pLysE on the minimal medium A. In the course of EMAP II cytokine expression the maximum amount of protein was observed if the inductor was added to the culture medium for the second hour of culture and the optimal concentration of the inducer of the synthesis IPTG was 1.25 mМ. The highest increase in expression of the target protein was observed in the culture of 4.5 h induction. A highly purified EMAP II cytokine was obtained to further construct its complex with the ligand and to study its characteristics. The recombinant EMAP II protein was titrated with dextran 70 solution. The change of fluorescence intensity of the protein was observed with increasing ligand concentration, which indicates the strong binding of the ligand to the recombinant protein and the formation of a stable complex. This indicates the stabilization of EMAP II structure in the complex, which is more resistant to high temperatures compared to free protein. Dynamic light scattering was used to study the effect of dextran 70 on the aggregation of the cytokine EMAP II. It was shown that the complex EMAP II with dextran 70 is quite strong and stable, the polysaccharide dextran 70 stabilizes the EMAP II protein globule and prevents aggregation processes. To understand the nature of the aggregates formation of EMAP II protein, we modeled the interaction between the spatial structures of the protein using Cluspro 2.0 and SymmDock web servers for the macromolecular docking. The analysis of spatial structures revealed that one of the key roles in the formation of contacts between protein molecules in the structure of protein-protein complexes was played by the unstructured loop 34DVGEIAPR41, which blocks the hydrophobic tryptophan pocket. In order to identify a potential dextran binding site of the surface of EMAP II, the flexible docking was performed using the AutoDock Vina software. According to models obtained in the binding of dextran 70 to EMAP II involved such residues as Arg12, Gly36, Glu37, Ile38, Arg41, Lys68, Lys71, Met72, Arg73, Leu76, Lys116, Asn119, Lys121, Lys123, Trp125, Lys166. When checking the nanocomposite preparation EMAP II with dextran 70 for the presence of lipolysaccharides, it was found that the preparation contained less than 0.5 IU/kg but more than 0.03 IU/kg. Toxicological testing of LD50 complex EMAP II with dextran 70 was performed on Balb/с laboratory mices. It was shown that after the infusion of the EMAP II complex with dextran 70 to mice at doses of 300 - 10 000 μg/kg no general toxic effect of the complex was not observed and it does not cause animal death. Using the PST cell culture (piglet testicles), it was shown that the complex of EMAP II with dextran 70 induces the production of TNF-α in the concentration range from 1.6 to 25.0 μg/ml. Interferon production by the cell cultures was not detected upon treatment with EMAP II complex with dextran 70. It was shown that dextran 70 does not affect TNF-α production. On the model of transplanted fragments of human prostate adenocarcinoma complex EMAP II with dextran 70 with a concentration of 10 μg/kg, it was found that the complex provided 77% inhibition of the tumor growth progress.

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