Expression of Chicken Liver 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase in Escherichia coli

Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain

“…Earlier work demonstrated that chicken liver 6PF-2K is activated by ATP, which reflects a negative co-operativity for binding of ATP to the enzyme [14,21]. Double-reciprocal plots of the 6PF-2K activity of CKB against ATP concentration clearly showed two slopes (results not shown), which yielded two K m values for ATP (Table 1).…”

“…The V max ratio of 6PF-2K to Fru-2,6-P # ase of CKB is approx. 18-fold greater than that of RKB [14]. The phosphorylation of RKB greatly reduces the affinity of the 6PF-2K for Fru-6-P by 10-15-fold [10].…”

“…The deletion mutants of CKB were engineered by replacing the EcoRI\BamHI fragment of wild-type CKB cDNA with that of corresponding deletion mutant forms of the separate bisphosphatase domain of CKB (CBD) [14,15]. All the site-directed mutations of CKB were generated by PCR using the Expand TM High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany).…”

“…All mutant forms of CKB and CBD were expressed in Escherichia coli BL21 (DE3) using the phage T7 RNA polymerase-based system, as described previously for wild-type CKB and CBD [14][15][16]. After induction with isopropyl β--thiogalactoside (Sigma ; 0.01 µM for CBD and 100 µM for CKB) at 22 mC for 24 h [17], the protein was extracted and purified using the procedure for CKB [14] or CBD [16] described previously.…”

“…After induction with isopropyl β--thiogalactoside (Sigma ; 0.01 µM for CBD and 100 µM for CKB) at 22 mC for 24 h [17], the protein was extracted and purified using the procedure for CKB [14] or CBD [16] described previously. All enzymes were purified to homogeneity, as judged by SDS\PAGE (results not shown).…”