Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum.
Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 10(7) cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (K(m)=1.7 microM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target.
Arya
Ranjana
R
Department of Biotechnology and Bioinformatics, Ranbaxy Research laboratories, Gurgaon, Haryana, India. ranjana.arya@ranbaxy.com
Aslam
Saima
S
Gupta
Shivani
S
Bora
Roop Singh
RS
Vijayakrishnan
Lalitha
L
Gulati
Pankaj
P
Naithani
Sudha
S
Mukherjee
Shohini
S
Dastidar
Sunanda
S
Bhattacharya
Alok
A
Saini
Kulvinder Singh
KS
D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000096:biosynthesis* / Q000302:isolation & purification; D004023:Dictyostelium / Q000201:enzymology* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D058988:Phosphodiesterase 4 Inhibitors; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013379:Substrate Specificity; D014170:Transformation, Genetic
D004791:Enzyme Inhibitors; D058988:Phosphodiesterase 4 Inhibitors; D011994:Recombinant Proteins; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517360:PDE4B protein, human