Expression of actin in Escherichia coli. Aggregation, solubilization, and functional analysis.

摘要: Wild type Dictyostelium discoideum actin (42 kDa) and a truncated form of were expressed in Escherichia coli. Amino-terminal sequencing indicated that the species was composed two peptides, which result internal translation initiation at Met-119 Met-123. After sonication or French press lysis, all present highly insoluble aggregates. When bacteria lysed directly into Sarkosyl detergent, most soluble, greater than 50% remained soluble after removed. Full-length wild purified using DNase I affinity chromatography gel filtration. This able both to polymerize bind myosin an ATP-sensitive manner, indicating it native. Affinity demonstrated bound same extent as synthesized eukaryotes, applicability this approach mutant forms actin. Thus, lysis procedures utilizing may prove useful isolating some other proteins are normally but become bacterial expression.