Purification and characterization of a recombinant pea cytoplasmic fructose-1,6-bisphosphatase.
Full-length cDNA encoding pea cytoplasmic fructose-1,6-bisphosphatase (cyFBPase) was cloned from a pea cDNA library. The cloned cDNA was introduced into the Escherichia coli expression vector pET-15b. The recombinant cyFBPase was expressed in E. coli BL21 (DE3) cells in a soluble form and purified to homogeneity by Ni(+)-NTA affinity chromatography. The identity of the recombinant cyFBPase was confirmed by SDS-PAGE and immunoblot analysis using a polyclonal anti-His tag antibody. The recombinant cyFBPase was active at neutral pH ranges (6.6-9.0) and thermostable as other cyFBPases. The activation energy (E(a)) and Arrhenius frequency factor were 17.4 kcal/mol and 2.6 x 10(12)/s, respectively. The K(M) and V(max) values of the recombinant enzyme were calculated as 10.47 microM and 109 micromol/min, respectively. In case of removal of histidine tag, the K(M) value was calculated as 5.03 microM. The recombinant enzyme was non-competitively and competitively inhibited by AMP and fructose-2,6-bisphosphate, respectively.
Jang
Hye-Kyung
HK
Plant Metabolism Research Center (PMRC) and Graduate School of Biotechnology, Kyung Hee University, Suwon 449-701, Republic of Korea.
Lee
Sang-Won
SW
Lee
Youn-Hyung
YH
Hahn
Tae-Ryong
TR
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D011994:Recombinant Proteins; D006597:Fructose-Bisphosphatase