Multigene families whose members show complex patterns of expression encode the subunits of myosin and actin in many organisms. This complexity has hampered genetic and biochemical analyses of individual members of these gene families. In order to map the functional domains of myosin and actin, several of their genes have been expressed in Escherichia coli and begun to assay their functions in vitro. In the past, many functional domains have been identified by partial proteolysis of native molecules. In contrast, bacterial expression affords a number of advantages to studying eukaryotic protein structure and function. One potential drawback of bacterial expression is the lack of posttranslational modifications in E. coli. However, this provides the opportunity to examine the effect of their absence on various functions. This chapter presents a number of methods for expressing and purifying myosin subunits and actin in E. coli. The issues discussed are: (1) vectors and quantities of protein expressed, (2) degradation and protease-deficient host strains, (3) internal initiation of transcription/translation, (4) growth conditions for expression strains, (5) solubility and lysis methods, and (6) purification schemes.
ASJC Scopus subject areas
- Molecular Biology