Purification and properties of genetically engineered protein containing the repeating sequence in the glycophorin-binding protein of malaria merozoite

RESUMO

A protein containing the glycophorin-binding sequence, M3R, has been genetically engineered in E. coli, and a method of its purification from the bacterial source has been established. The method involvers a) extraction, b) heat treatment at 80' for 3 min, c) concentration of M3R by acid precipitation, d) HPLC on a reverse-phase C8 column, and e) purification by reverse-phase C4 chromatography. The purified protein migrates as a single band of Mw=22000. M3R has been assayed by the rabbit antibody raised against a synthetic peptide containing a partial sequence of the protein. The overall yield has been approximately 20 mg of pure protein from 100 gm of bacterial paste. Automated sequence analysis has confirmed the purity as well as the identity of the protein as M3R; its sequence of 15 residues from the NH2-terminus agrees with that predicted from the gene sequence. Structural analyses of its peptide fragments have further confirmed correctness of its sequence. Rabbit antibody prepared against M3R has been found to react with the merozoite of P.falcipurm merozoite