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Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum
Author(s) -
Arya Ranjana,
Aslam Saima,
Gupta Shivani,
Bora Roop Singh,
Vijayakrishnan Lalitha,
Gulati Pankaj,
Naithani Sudha,
Mukherjee Shohini,
Dastidar Sunanda,
Bhattacharya Alok,
Saini Kulvinder Singh
Publication year - 2008
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200700256
Subject(s) - dictyostelium discoideum , recombinant dna , phosphodiesterase , affinity chromatography , high throughput screening , mycetozoa , biochemistry , chemistry , biology , microbiology and biotechnology , enzyme , gene
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 μM). 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.