Structure-Function studies based on in vitro expression

Abstract

The advent of efficient procedures for site-directed and random in vitro mutagenesis has led to detailed exploration of protein structure-function relationships. Using these approaches, amino acid residues critical for protein activity can be determined in the absence of X-ray crystallographic data (1, 2). An important prerequisite for any study examining protein structure-function relationships is an efficient expression system that gives not only adequate protein yield, but also fully native protein. Expression of mammalian genes in E. coli is often very successful with good protein yields, but some proteins are not correctly folded and, as a consequence, are not fully biologically active. Structure-function studies can be hampered by poor expression of modified proteins in both yeast and E. coli (3). Mammalian expression systems can give high-fidelity protein, but often the protein yield is poor and significant purification may be required. Systematic studies of protein using mutagenesis often require the analysis of a large number of modified proteins. If this process requires recloning into expression vectors, expression of protein, and purification, the analysis of a large number of proteins becomes prohibitive. The rabbit reticulocyte lysate (RRL,) translation system can be used to provide small quantities of protein when primed with synthetic mRNA generated in vitro from DNA templates with bacterial RNA polymerases.