Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:2.3.1.28 (
chloramphenicol acetyltransferase
)
5,100
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
A 19-kilobase BamHI fragment encoding the recB (
exonuclease V
), recC (
exonuclease V
), ptr (protease III), thyA, and argA genes of Escherichia coli K-12 was cloned into a multicopy plasmid (pCDK3). In E. coli maxicells, the plasmid specified the synthesis of seven polypeptides of 140,000 (recC), 128,000 (recB), 110,000 (ptr), 53,000 (argA), 50,000, 33,000 (thyA), and 22,000 Mr, as well as beta-lactamase and
chloramphenicol acetyltransferase
. From analysis of subclones and Tn1000 insertions, it appears that the 110,000- and 50,000-Mr proteins originated from the ptr DNA coding sequence which is located between the recB and recC genes. Although recC, ptr, and recB were physically closely linked and transcribed in the same direction, they do not appear to constitute an operon. Cells carrying pCDK3 contained a 30- to 50-fold increase in
exonuclease V
activity, without affecting cell viability.
...
PMID:Physical and biochemical analysis of the cloned recB and recC genes of Escherichia coli K-12. 631 51
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.
...
PMID:Increasing PCR fragment stability and protein yields in a cell-free system with genetically modified Escherichia coli extracts. 1625 43
