Expression of actin in Escherichia coli. Aggregation, solubilization, and functional analysis.
Wild type Dictyostelium discoideum actin (42 kDa) and a truncated form of actin were expressed in Escherichia coli. Amino-terminal sequencing indicated that the truncated species was composed of two peptides, which were the result of internal translation initiation at Met-119 and Met-123. After sonication or French press lysis, all of the actin was present in highly insoluble aggregates. When bacteria were lysed directly into Sarkosyl detergent, most of the actin was soluble, and greater than 50% remained soluble after Sarkosyl was removed. Full-length wild type actin was purified using DNase I affinity chromatography and gel filtration. This species was able both to polymerize and to bind myosin in an ATP-sensitive manner, indicating it was native. Affinity chromatography demonstrated that the truncated form of actin bound DNase I to the same extent as actin synthesized in eukaryotes, indicating the applicability of this approach to mutant forms of actin. Thus, lysis procedures utilizing Sarkosyl may prove useful in isolating some of the other proteins which are normally soluble but become insoluble after bacterial expression.
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461.
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Frankel
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Condeelis
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Leinwand
D000199:Actins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009218:Myosins / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure
D000199:Actins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D003850:Deoxyribonuclease I; D009218:Myosins