17] Synthesis and expression of synthetic genes: Applications to structure-function studies of receptors

Abstract

Publisher Summary This chapter discusses synthesis and expression of synthetic genes. While a number of methods are available for site-directed mutagenesis, the use of a properly designed synthetic gene offers many advantages, particularly where extensive mutagenesis is planned. A synthetic gene used for heterologous expression codes for the correct amino acid sequence but contains an artificial nucleotide sequence. The nucleotide sequence is generally designed to contain a large number of unique restriction endonuclease cleavage sites. The degeneracy of the genetic code and the large number of available restriction enzymes allows on the order of 40 unique restriction sites to be introduced for a synthetic gene of 1-kb pairs in length. Gene synthesis has been particularly useful in the study of G protein-coupled receptors. The superfamily of G protein-coupled receptors consists of a large number of related seven-transmembrane segment plasma membrane proteins. In studying receptor function by heterologous expression of mutant receptors, it is often useful to create chimeric receptors in which putative functional domains are exchanged.