[18] Analysis of G-protein α and βγ subunits by in vitro translation

Publisher Summary This chapter discusses analysis of G-protein α and βγ subunits by in vitro translation. The analysis of G-protein subunits that have been modified by point mutation, deletion, or formation of chimeras requires that the altered protein be expressed. Cell-free transcription and translation of modified cDNAs offer certain advantages and disadvantages compared to various cellular systems. An important disadvantage is that cell-free translation yields only a small amount of synthesized protein that is generated in a concentrated mixture of many cellular proteins. It would, therefore, be very difficult to purify the small amount of specific protein from the very large amount of cellular components. This disadvantage is counterbalanced by the ability to radiolabel the specific protein selectively. Because only specific mRNA is added to the in vitro translation mixture, the desired protein usually represents the only radioactive band produced. Thus, if methods exist to evaluate the structure and function of the radioactive protein, without purifying it from the nonradioactive proteins present, then in vitro translation offers a rapid and attractive way of screening the consequences of modifications introduced into proteins. The chapter discusses techniques that can be used to assay the function of G-protein α, β, and γ subunits that have been synthesized in vitro. The modifications that have been made to standard in vitro transcription-translation systems are reviewed in order to enhance the yield of G-protein subunits.