Synthesis of the ensembles from succinylated interleukin-2 derivatives and their biological activity in vitro
DOI:
https://doi.org/10.15587/2313-8416.2015.53985Keywords:
Agilent-2100, bioactivity, succinylated interleukin-2, high-performance liquid chromatography, recombinant interleukin-2Abstract
Purpose: The biological activity (direct anticancer action in vitro) of combinatorial IL-2 succinylated derivatives was examined. Acylation of IL-2 was performed using succinic anhydride with various levels of acylation, with the formation of a complex assembly of many derivatives (self-assembled quasi-living structure).
Methods: In the study, we used recombinant interleukin-2 (IL-2) (Ronkoleukin, Russia) in oxidizing form and other reagents from Sigma-Aldrich and Fluka (USA). For acylation, recombinant IL-2 in the form of a matrix fluid with a protein concentration of 0.6 mg/ml was used. The IL-2 matrix solution was brought to a pH of 8.0 through the addition of a 0.01% solution of sodium hydroxide. The synthesized ensemble of succinyl-IL-2 was analyzed using the capillary gel electrophoresis method in an Agilent-2100 bioanalyzer. The molecular masses of the synthesized ensemble were established in comparison to standard samples of low-molecular proteins with known molecular masses that were part of the bioanalyzer’s collection. The additional negative charge of the modified IL-2 was determined by a FPLC (Fast Protein Liquid Chromatography) system from Pharmacia (Woerden, Netherlands). CTLL-2, a murine IL-2-dependent cell line, was obtained fromBiokontrol (Kiev, Ukraine). These respondingcells were used in proliferative assays comparing the activityof the IL-2 and succinylated IL-2 derivatives to the WHO InternationalStandard. For statistical data analysis, the one-way ANOVA was used.
Results: After treatment with Suc-IL-2, BTL were studied in vitro on the CTLL-2 cell line, dose-dependently, in a BTL induction reaction. The highest level of biological activity in acylated IL-2 was observed in Suc-IL-2 with four modified lysine residues. In conclusion, succinylated IL-2 with four substituted lysines may be useful as a prospective anticancer agent.
Conclusions: Ten-fold increase in the biological activity of IL-2 was observed after partial succinylation. This phenomenon can be used in the further IL-2 drugs biotechnological development for increasing the concentration of the main active substance in medicinal form
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