Analysis of M13 Procoat Assembly into Membranes in Vitro

This chapter analyzes the M13 procoat assembly into membranes in vitro. The major coat protein of the coliphage M13 becomes a transmembrane component of the inner Escherichia coli membrane prior to its incorporation into new virus particles. It has proved to be useful in the studies of membrane protein assembly in vitro. The major products following the addition of either MI3 replicative form (RF-I) DNA or messenger RNA (mRNA) isolated from Ml3-infected E. coli to a cell-free transcription-translation reaction are the products of gene 5 and gene 8. The analysis of the association of newly synthesized proteins with both inner membrane vesicles and reconstituted vesicles involves measuring the extent of processing of precursor forms by the vesicles and localizing the proteins with respect to the plane of the lipid bilayer. The extent of in vitro conversion of precursors to the mature form is assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the labeled proteins and fluorography of the dried gel. In the studies of the assembly of M13 procoat in vitro, it is useful to employ a variety of proteinase concentrations. Following digestion all the remaining labeled proteins by are analyzed by gel electrophoresis. The proteinase concentrations was determined that degraded peripherally bound substrates, such as the product of gene 5, compared with procoat and coat protein.