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Название: Two-Hybrid System Methods and Protocols
Автор: MacDonald P.
Many, if not all, essential biological processes require selective interactions
between proteins. Complex signaling systems require sequential, ordered
protein–protein interactions at essentially all levels of the signaling cascade. For
example, peptide hormones interact with selective membrane receptor proteins,
and autophosphorylation of the receptor then recruits other key regulatory
proteins that initiate kinase cascades in which each phosphorylation event
requires selective recognition of the protein substrate. The ultimate signaling
effect, in many cases, is the regulation of RNA polymerase II-directed transcription
in the nucleus, a process that involves numerous, multiprotein complexes
important for transcription initiation, elongation, termination, and reinitiation.
Defining, characterizing, and understanding the relevance of these protein–
protein interactions is an arduous task, but substantial inroads have been made
over the past 20 years. The development of more recent methodologies, such as
mammalian expression systems, immunopurification schemes, expression
cloning strategies, surface plasmon resonance (BiaCore), and nanosequencing
technologies, has contributed a wealth of new insights into these complex
multiprotein mechanisms and clearly accelerated the discovery process.
Arguably, the yeast two-hybrid system has been one of the predominant and most
powerful tools in this discovery process.