Expression of rat liver fructose-1,6-bisphosphatase in Escherichia coli.
Rat liver fructose-1,6-bisphosphatase was expressed in Escherichia coli using a T7 RNA polymerase-transcribed expression system. Maximum yields of soluble active enzyme were obtained when the bacterial host cell, BL21(DE3), carrying the expression plasmid was grown and transcription induced in LB medium at 37 degrees C. Approximately 20mg of fructose-1,6-bisphosphatase are synthesized per liter of culture after 4hr, of which about 10mg are soluble and enzymatically active. Expressed fructose-1,6-bisphosphatase, purified to homogeneity by substrate elution from a carboxymethyl Sephadex column, was indistinguishable from that purified from rat liver in terms of subunit size and kinetic properties. The in vitro expression of fructose-1,6-bisphosphatase in an heterologous system is a necessary preliminary step for future studies on site-directed mutant enzyme forms.
el-Maghrabi
M R
MR
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
Pilkis
S J
SJ
D000818:Animals; D001483:Base Sequence; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D006597:Fructose-Bisphosphatase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D008715:Methionine; D006597:Fructose-Bisphosphatase