This chapter discusses transient transfection assay procedure of the herpesvirus proteinase. It describes the methods that are used to prepare plasmids, carry out transfections, separate proteins by SDS–PAGE, and identify proteinase and substrate proteins by western and immunoprecipitation immunoassays. Human embryonal kidney (HEK) cells are transfected with a plasmid encoding the proteinase, alone or together with a plasmid encoding a substrate, the transfected cells are then harvested 2 or 3 days after adding the DNAs and the transfected-cell proteins are resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the proteins of interest are visualized by Western immunoassays. Proteolytic cleavages are monitored by the disappearance of the precursor forms of the proteins and appearance of the appropriate product forms. These proteins are identifiable by their migration following SDS–PAGE and by their reactivity with specific antisera. The eukaryotic expression vector RSV.5(neo) is used to express wild-type and mutant forms of cytomegalovirus assembling, its precursor, pNP1, and its substrate, pAP. Mutations are verified by dideoxynucleotide sequence analysis.
ASJC Scopus subject areas
- Molecular Biology