Low concentration of inducer favors production of active form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli.
Expression of chicken and rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, in Escherichia coli encountered two common problems: the chicken enzyme was liable to proteolysis and the rat enzyme was prone to form inclusion bodies. Reducing the rate of protein synthesis by lowering either growth temperature or isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration alleviated these two problems. Growth at 22 degrees C was optimum for expression of both enzymes. The optimum range of IPTG concentration for expression was 0.1-1 microM for the chicken liver bifunctional enzyme and 10 microM for rat liver enzyme. The components of growth medium also influenced the production. Compared with Luria-Bertani medium, an enriched medium-tryptone-phosphate medium-tripled the production of the active enzymes. Addition of glucose (0.2%) doubled the expression level of active chicken liver enzyme, but reduced the production of active rat liver enzyme to half the maximal level, while the phosphate in tryptone-phosphate medium had no effect on the production of the two enzymes.
Yang
Q H
QH
Shanghai Institute of Biochemistry, Academia Sinica, China.
Wu
C L
CL
Lin
K
K
Li
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L
D000818:Animals; D002645:Chickens; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005947:Glucose / Q000494:pharmacology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D010770:Phosphotransferases / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature
D003470:Culture Media; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D005947:Glucose