Biochemical and functional characterization of a new recombinant phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum

Phospholipase A2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA2inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI 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 recγCdcPLI 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. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately β-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA2 from honeybee (Kd = 1.48 μM) and was able to inhibit the PLA2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA2 activity.
Keywords
PLA2 inhibitor;  Antiangiogenic;  Recombinant protein

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Gimenes SNC, Aglas L, Wildner S, Huber S, Silveira ACP, Lopes DS, et al. Biochemical and functional characterization of a new recombinant phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum. Int. J. Biol. Macromol.. 2020 Dec;164:1545-1553. doi:10.1016/j.ijbiomac.2020.07.221.
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