Specific antibodies against binase: Preparation and application

V.I. Vershinina^*, E.V. Dudkina^**, V.V. Ulyanova^***, V.A. Kalashnikov^****, O.N. Ilinskaya^*****

Kazan Federal University, Kazan, 420008 Russia

E-mail: ^*valver1951@mail.ru, ^**lenatimonina@rambler.ru, ^***ulyanova.vera@gmail.com, ^****icebuldogq@gmail.com, ^*****Ilinskaya_kfu@mail.ru

Received September 6, 2021

ORIGINAL ARTICLE

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DOI: 10.26907/2542-064X.2021.4.557-568

For citation: Vershinina V.I., Dudkina E.V., Ulyanova V.V., Kalashnikov V.A., Ilinskaya O.N. Specific antibodies against binase: Preparation and application. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2021, vol. 163, no. 4, pp. 557–568. doi: 10.26907/2542-064X.2021.4.557-568. (In Russian)

Abstract

Secreted ribonuclease of Bacillus pumilus (binase) is a promising antitumor agent with established selective cytotoxicity against various types of cancer cells expressing specific oncogenes. However, molecular mechanisms behind the apoptosis-inducing action of binase remain unclear. It was shown that catalytic activity, protein charge, oligomerization, and interaction with RAS oncogene contribute to binase cytotoxicity. To identify the intracellular targets of binase antitumor activity, binase-specific molecular tools could be of great assistance. In this article, a specific polyclonal rabbit serum was obtained by rabbit immunization with the industrial binase preparation using four subcutaneous protein injections with Freund adjuvant and revaccination. Isolation and purification of antibodies against ribonuclease from the serum were performed using affinity chromatography. Affinity resin was prepared by the immobilization of binase in CNBr-activated Sepharose 4. The specificity of the serum and antibodies was confirmed by agar gel immunodiffusion assay and western blotting. It was demonstrated that the antibodies can be successfully applied for testing native and inactivated forms of binase, as well as binase within cell lysates and protein complexes. The obtained specific antibodies will help to reveal the localization of binase in eukaryotic cells. They will also be useful for determining binase interaction with surface and intracellular targets, including viral components.

Keywords: ribonuclease, binase, antitumor activity, obtaining of specific antibodies

Acknowledgements. This study was performed as part of the Kazan Federal University Strategic Academic Leadership Program and supported by the Russian Science Foundation (project no. 21-74-10036).

Figure Captions

Fig. 1. The immunodiffusion reaction in agar gel. Peripheral wells – immune serum from different rabbits after the third (a) and fifth injection (b), central wells (Bi) – binase solution (1 mg/mL).

Fig. 2. The precipitation reaction of the protein complex binase-barstar by specific antibodies against binase. Bi – binase, Bi+Brst – binase in a complex with a specific inhibitor barstar, C – serum to binase. The reaction demonstrates the ability of antibodies against binase to detect the enzyme in the protein complex.

Fig. 3. Control of antibodies extraction from the serum during chromatography and in the precipitation reaction: 1 – initial serum, 2 – serum after the sorption of antibodies, 3 – flow-through containing unbound antibodies; Bi – binase.

Fig. 4. Analysis of the specificity of the obtained antibodies by western blotting: 1 – native binase; 2 – recombinant inactive binase isolated from the periplasm of E. coli BL21 pML163-H101E; 3 – recombinant inactive binase in the periplasmic fraction of E. coli BL21 pML163-H101E; 4 – lysozyme.

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