A novel cell-free protein synthesis system.
An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.
Sitaraman
Kalavathy
K
SAIC-National Cancer Institute at Frederick, 1050 Boyles Street, Building 327, Frederick, MD 21702-1201, USA.
Esposito
Dominic
D
Klarmann
George
G
Le Grice
Stuart F
SF
Hartley
James L
JL
Chatterjee
Deb K
DK
D010582:Bacteriophage lambda / Q000737:chemistry; D001709:Biotechnology; D002458:Cell Fractionation; D002474:Cell-Free System; D004268:DNA-Binding Proteins; D004734:Energy Metabolism; D004926:Escherichia coli / Q000378:metabolism; D005988:Glyceric Acids / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D013698:Templates, Genetic; D013997:Time Factors; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D005988:Glyceric Acids; D011506:Proteins; D014764:Viral Proteins; C065410:gam protein, Coliphage; D010728:Phosphoenolpyruvate; C005156:3-phosphoglycerate