Purification of recombinant human phosphodiesterase 7A expressed in Dictyostelium discoideum.
Phosphodiesterase plays an important role in regulating inflammatory pathways and T cell function. The development of phosphodiesterase 7 inhibitor may give better efficacy profile over phosphodiesterase 4 inhibitors. However, the recombinant phosphodiesterase 7 is required in large quantity for high-throughput screening of new drugs by in vitro enzymatic assays. In the present study, recombinant human PDE7A1 was expressed in Dictyostelium discoideum under the control of constitutively active actin-15 promoter. The cytosolic localization of the expressed protein was confirmed by immunofluorescence studies. Upto 2 mg of recombinant protein was purified using His-Tag affinity column chromatography followed by ion-exchange Resource Q column purification. The recombinant protein expressed in D. discoideum followed Michaelis-Menten kinetics similar to the protein expressed in mammalian system and showed no major changes in affinity to substrate or inhibitors. Thus, our study clearly demonstrates a robust expression system for successful bulk production of pharmacologically active isoform of human PDE7A1 required for high-throughput assays.
Arya
Ranjana
R
Room No. 127, School of Biotechnology, Jawaharlal Nehru University, New Mehrauli Road, New Delhi-110067, India. ranjanaa@mail.jnu.ac.in
Gupta
Shivani
S
Aslam
Saima
S
Kaur
Namrata Jit
NJ
Seth
Aayush
A
Eapen
Mathew S
MS
Malik
Renu
R
Vijayakrishnan
Lalitha
L
Saini
Kulvinder Singh
KS
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology; D054708:Cyclic Nucleotide Phosphodiesterases, Type 7 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D004023:Dictyostelium / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D000242:Cyclic AMP; D054708:Cyclic Nucleotide Phosphodiesterases, Type 7; C517458:PDE7A protein, human