31] Expression and mutagenesis of the regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

Abstract

Publisher Summary The advancement of technology has provided new dimensions for asking questions about protein structure and function, about the role that any given protein plays, and about the regulation of its expression. Questions of expression now can be addressed directly. Of fundamental importance will be how the expression of R and C subunits is coordinated. With at least one R subunit gene cloned, it is possible to selectively mutate various regions of the regulatory subunit to better understand those specific structural features that convey functional properties to the protein. Following isolation of the cloned gene, the next step in this process is the construction of an expression vector. In addition to being a step on the pathway toward mutagenesis, the expression of R subunit in bacteria also can provide a mechanism for rapidly producing large amounts of protein. This chapter describes the construction of such expression vector. Because the R subunit is not thought to be subject to major posttranslational modification, such as glycosylation, the host of choice is bacteria, and a wide variety of vectors are available for designing constructs that will lead to the expression of a given protein.