Ponente
Descripción
CogiTx1 is a defensing-like subtilisin inhibitor isolated from the sea anemone C. gigantea, the in-depth functional characterization of this molecule needs to be completed, since the purification from the natural source has a very low yield, a source of pure and well-folded protein is needed. Therefore, the aim of this work was to obtain two recombinant variants of CogiTx1. To achieve this goal, the sequence of CogiTx1 variants, optimized for expression in Escherichia coli, including a TEV protease site, were inserted in the plasmid (pET32a/CogiTx1r and pET32a/CogiTx1+GR) and expressed in Shuffle E. coli cells. Expression of the fusion proteins Trx-TEV-CogiTx1 was achieved by IPTG induction. The fusion proteins were purified by Ni-NTA chromatography, dialyzed against TEV protease buffer, concentrated in 10kDA membranes and digested with TEV to release the CogiTx1r/CogiTx1+GR. The reaction mixture was applied to a Hiload Superdex 75 16/60 matrix to obtain the free CogiTX1r/CogiTx1+GR. Purity of the proteins was checked by SDS-PAGE 15%, the sequence was checked by mass spectrometry and the subtilisin inhibitory activity evaluated using the Suc-AAPF-pNA substrate. CogiTx1r and CogiTx1+GR are well-folded peptides with 4970 and 5190 Da respectively and both molecules present the expected amino acid sequence. CogiTx1r is active against subtilisin, porcine pancreatic elastase, proteinase K and Stenothophomonas maltophilia protease 1 (rStmPr1). However, CogiTx1+GR (sequence derived from the cDNA) is not active against any of the enzymes evaluated, result that could be due to the presence of two extra residues –GR at the C-terminal when compared with CogiTx1r (sequence of the natural protein). In conclusion, the expression in E. coli of CogiTx1 variants was successful and the results suggest that the modification observed in the natural protein is important for the activity of CogiTx1.
