Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N.  oxiana

Publication date: February 2017 Source:Protein Expression and Purification, Volume 130 Author(s): M.A. Shulepko, E.N. Lyukmanova, Z.O. Shenkarev, P.V. Dubovskii, M.V. Astapova, A.V. Feofanov, A.S. Arseniev, Y.N. Utkin, M.P. Kirpichnikov, D.A. Dolgikh Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the ‘three-finger’ protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60–90 residues) with high disulfide bond content (4–5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of 13C,15N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different...
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research