Expression of Escherichia coli homoserine kinase in mouse 3T3 cells.
The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.
Rowett Research Institute, Scotland, U.K.
W D
WD
Rees
S M
SM
Hay
H J
HJ
Flint
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006714:Homoserine / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D012313:RNA / Q000032:analysis; D014162:Transfection
D012313:RNA; D006714:Homoserine; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021370:homoserine kinase