title: Expression of native rabbit light meromyosin in Escherichia coli. Observation of a powerful internal translation initiation site.
abstract: The cDNA-sequence coding for rabbit skeletal muscle light meromyosin (LMM) was placed under the control of the lambda promoter (PL) of an Escherichia coli expression vector. The resulting plasmid pEXLMM74 expressed non-fused rabbit skeletal muscle LMM with yields ranging from 1 to 5% of the total proteins of E. coli. This LMM was specifically recognized by polyclonal antibodies raised against chicken pectoralis muscle myosin. It could be highly enriched from E. coli extracts by using two cycles of high and low ionic strength buffer. The partially purified protein contained a major side-product, with a calculated molecular mass of 59 kilodaltons, that is produced by translation initiation from a site in the coding region of LMM. After deletion of the translation initiation site derived from the expression plasmid, only the 59 kilodalton protein is expressed in E. coli from the resulting plasmid pEXLMM59. Both the 74 and 59 kilodalton proteins were shown to form paracrystals. They were studied by electron microscopy using negative staining and were found to show characteristic striations with an axial periodicity of about 43 nm. By circular dichroism measurement we showed that the purified 59 kilodalton protein is folded mostly as an alpha-helix.
authors.affiliation: Max-Planck-Institut für medizinische Forschung Abteilung Biophysik, Heidelberg, F.R.G.
authors.forename: K
authors.initials: K
authors.lastname: Maeda
authors.forename: G
authors.initials: G
authors.lastname: Sczakiel
authors.forename: W
authors.initials: W
authors.lastname: Hofmann
authors.forename: J F
authors.initials: JF
authors.lastname: Menetret
authors.forename: A
authors.initials: A
authors.lastname: Wittinghufer
mesh_terms: D000818:Animals; D001483:Base Sequence; D004270:DNA, Circular / Q000235:genetics; D004926:Escherichia coli; D005786:Gene Expression Regulation; D015879:Myosin Subfragments / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D011817:Rabbits
chemical_list: D004270:DNA, Circular; D015879:Myosin Subfragments