Soluble expression in Escherichia coli of active human cyclic nucleotide phosphodiesterase isoform 4B2 in fusion with maltose-binding protein.
Recombinant expression in Escherichia coli of human cyclic nucleotide phosphodiesterase 4B2 (hPDE4B2) fused to maltose-binding-protein (MBP-hPDE4B2) was investigated. hPDE4B2 DNA amplified via nested RT-PCR with total RNAs from U937 cells was ligated with pMAL-p2x. After induction at 18 degrees C for 16 h, soluble MBP-hPDE4B2 was produced in E. coli. MBP-hPDE4B2 after amylose-resin chromatography showed 35% homogeneity, and its Michaelis-Menten constant was 10+/-2 microM (n=3). Rolipram had a dissociation constant of 9+/-2 nM (n=2), and zinc ion was a potent inhibitor. Hence, MBP-hPDE4B2 was expressed in E. coli as a soluble active protein.
Zhu
Sha
S
Chongqing Key Laboratory of Biochemical and Molecular Pharmacology, Chongqing Medical University, Chongqing 400016, China.
Yang
Genqing
G
Yang
Xiaolan
X
Zhao
Yunsheng
Y
Li
Xiang
X
Deng
Ping
P
Xie
Yanling
Y
Gan
Zhiyong
Z
Liu
Yin
Y
Li
Zhirong
Z
Liao
Juan
J
Yu
Ming'an
M
Liao
Fei
F
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D002457:Cell Extracts; D045744:Cell Line, Tumor; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D002352:Carrier Proteins; D002457:Cell Extracts; D007527:Isoenzymes; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4