Biochemical and Functional Characterization of a New Recombinant Phospholipase A2 Inhibitor from Crotalus Durissus Collilineatus Snake Serum.

Phospholipase A(2) plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA(2) activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of gamma CdcPLI, the natural PLA(2) inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the gamma CdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant rec gamma CdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. Rec gamma CdcPLI showed similar secondary structure in CD and FTIR, revealing predominately beta-strands. Analogous to the natural protein, rec gamma CdcPLI was able to form oligomers of similar to 5.5 nm. The inhibitor was efficiently binding to PLA(2) from honeybee (Kd = 1.48 mu M) and was able to inhibit the PLA(2) activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of rec gamma CdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA(2) activity. (C) 2020 Elsevier B.V. All rights reserved.