10] Cell-free expression vector: Use of insect virus translational initiation signal for in vitro gene expression

Abstract

Publisher Summary This chapter discusses the use of insect virus translational initiation signal for in vitro gene expression. Gene expression in a cell-free system avoids various cellular regulatory mechanisms that often pose serious problems for in vivo gene expression. Some of these problems are (1) rapid degradation of the expressed gene products by host enzymes, (2) the adverse effect of the foreign protein on the normal host metabolism, and (3) sensitivity of the heterologous RNA transcripts to various cellular regulatory controls, such as splicing and transportation into the cytoplasm. In addition, cell-free gene expression makes it possible to study the expressed gene products without their prior purification from host cell proteins of similar structure and function. Cell-free expression is limited, however, to the use of genes that carry their own signals to initiate translation efficiently. Translational initiation signals are contained within sequences in the 5´ untranslated region (UTR) as well as in the vicinity of the initiator ATG of a eukaryotic mRNA, where ribosomes bind and function to initiate protein synthesis.