Rapid expression of functional genomic libraries.
Genomic-scale analysis of protein function is currently limited by the ability to rapidly express the enormous diversity of protein targets in their active form. We describe a method to construct transcriptionally active expression templates (ETs) in parallel using a single PCR step wherein the overlap-extension reaction for addition of transcription regulatory elements is separated from the amplification of the full-length product by using a GC-rich single primer. Over 90% of 55 diverse genomic targets were extended with T7 regulatory elements to form ETs in high yield and purity. The unpurified ETs directed protein expression using a cell-free protein synthesis (CFPS) system supplemented with cofactors and metal ions to activate a variety of enzymes. Higher activities were obtained in the modified CFPS reactions compared to standard reaction conditions. Protein purification was avoided because the expressed enzyme activity was significantly greater than the background activity associated with the cell extract. These improvements in the parallel synthesis of linear ETs combined with enhanced in vitro enzyme activation help to make CFPS systems more attractive platforms for high-throughput evaluation of protein function.
Woodrow
Kim A
KA
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Airen
Isoken O
IO
Swartz
James R
JR
D017123:Bacteriophage T7 / Q000235:genetics; D002474:Cell-Free System; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D016680:Genome, Bacterial / Q000235:genetics*; D015698:Genomic Library; D016366:Open Reading Frames / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D029968:Escherichia coli Proteins