[27] Isolation and properties of protein factors involved in polypeptide chain initiation in Escherichia coli

This chapter describes a method that yields highly purified initiation factors, and free of transfer factor or nucleoside triphosphatase contamination, in Escherichia coli.

Abstract

Publisher Summary This chapter describes a method that yields highly purified initiation factors, and free of transfer factor or nucleoside triphosphatase contamination. The initiation of protein synthesis in Escherichia coli proceeds with the formation of a complex consisting of mRNA, a 70 S ribosome, and the specific initiator, formylmethionyl-tRNA (fMet-tRNA). Formation of the initiation complex requires GTP, Mg 2+ , and NH 4+ ions and, in addition, several protein factors that are loosely associated with ribosomes and can be dissociated by high salt treatment. The purification procedure for the initiation factors involves preliminary separation of initiation factors on DEAE-Cellulose, followed by further purification of initiation factors FI, FII, and FIII by various types of chromatography. FI is a heat-stable, basic protein consisting of a single polypeptide chain of molecular weight 9000. FII is also a basic protein, based upon its elution from CM-Sephadex and its migration in gel electrophoresis, and consists of a single polypeptide chain of molecular weight 21,000, while FIII is an acidic, heat-labile protein, and polyacrylamide gel electrophoresis of FIII phosphocellulose fractions reveals 1-4 protein bands, depending upon the position of elution from the phosphocellulose column.