Preparation of nitrophorin 7 (Δ1-3) from Rhodnius prolixus without start-methionine using recombinant expression in< i> Escherichia coli.

The heterologous recombinant expression of proteins in Escherichia coli without start methionine is a common problem. The nitrophorin-7 heme properties and function strongly depend on the accurate N-terminal amino acid sequence. Leading protein expression into the periplasm by fusion with the leader peptide pelB yields functional protein; however, the folded protein sticks to the cell debris. Therefore, the periplasmic fraction was dissolved in guanidinium chloride and folded by a drop-in method. Separation from impurities including residual pelB-nitrophorin-7 required to establish an unconventional chromatographic technique using calcium loaded Chelating Sepharose as cation exchanger and elution by a linear Ca... More