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A sequential expression system for high‐throughput functional genomic analysis
Author(s) -
Woodrow Kim A.,
Swartz James R.
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700471
Subject(s) - genomic dna , nucleic acid , gene , computational biology , dna , plasmid , template , reporter gene , biology , gene expression , expression vector , biochemistry , microbiology and biotechnology , recombinant dna , computer science , programming language
A method employing sequential rounds of cell‐free protein synthesis (CFPS) was developed to identify gene products influencing the complex metabolic systems that result in protein accumulation and folding in vitro . The first round of CFPS creates an array of cell extracts individually enriched with a single gene product expressed in‐parallel from linear DNA expression templates (ETs). The cell extract is engineered to enhance template stability and to provide reaction conditions conducive for general protein activation. Following first‐round expression, linear templates are selectively degraded and a plasmid template for a reporter enzyme is added to initiate a subsequent round of protein expression. Reporter concentration and activity identify first‐round gene products that affect amino acid and nucleic acid stability, energy supply, protein expression, stability, and activation. This sequential CFPS system provides a unique format for the functional genomic identification of broadly diverse metabolic activities.