Increasing PCR fragment stability and protein yields in a cell-free system with genetically modified Escherichia coli extracts.
Escherichia coli cell-free protein synthesis is a highly productive system that can be applied to high throughput expression from polymerase chain reaction (PCR) products in 96-well plates for proteomic studies as well as protein evolution. However, linear DNA instability appears to be a major limitation of the system. We modified the genome of the E. coli strain A19 by removing the endA gene encoding the endonuclease I and replacing the recCBD operon (in which recD encodes the exonuclease V) by the lambda phage recombination system. Using the cell extract from this new strain increased the stability of PCR products amplified from a plasmid containing the cat gene. This resulted in CAT (chloramphenicol acetyltransferase) production from PCR products comparable to that from plasmids (500-600 microg/ml) in a batch reaction. We show that cell-free protein synthesis reactions using PCR products amplified from genomic DNA and extended with the T7 promoter and the T7 terminator give the same high yields of proteins (550 microg/ml) in 96-well plates. With this system, it was possible to rapidly express a range of cytoplasmic and periplasmic proteins.
Michel-Reydellet
Nathalie
N
Department of Chemical Engineering, Stanford University, Stanford, Calif. 94305-5025, USA.
Woodrow
Kim
K
Swartz
James
J
D001426:Bacterial Proteins / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D043211:Exodeoxyribonuclease V / Q000235:genetics; D017353:Gene Deletion; D017930:Genes, Reporter; D005818:Genetic Engineering; D008565:Membrane Proteins / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D045522:Recombinases / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D045522:Recombinases; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D004706:Endodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C064668:endA protein, bacteria