Molecular Cloning And Heterologous Expression Of Human Phosphodiesterase 4B2 Gene

Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) through hydrolysis of these second messengers. Phosphodiesterase 4 (PDE4) is a family of cAMP-specific PDE expressed by immune and inflammatory cells, including neutrophils, T-lymphocytes, macrophages, and eosinophils. Because of their great market potential and therapeutic importance, PDE inhibitors became recognized as important therapeutic agents in the treatment of various diseases. Phosphodiesterase-4 selective inhibitors have therapeutic potential for treating major diseases such as asthma and chronic obstructive pulmonary disease, as well as depression, Parkinson's disease and Alzheimer's disease. In this paper, human phosphodiesterase 4B2 gene was cloned and expressed in the E. coli and yeast cells. The expressed protein will be used to set up a high throughput screening (HTS) model for the screening of their inhibitors.Total RNA was isolated from the of the THP-1 cells (human leukemic monocyte cell line) which were incubated with 1ng/ml LPS for 3h at 37℃. The cDNA of human PDE4B2 was amplified by RT-PCR with the total RNA as a template. DNA sequencing and BLAST results indicated that there were three nucleotides different from the reported sequence in GenBank (accession number M₉7515). Using overlap extension, the cDNA was site-directed mutated to the correct sequence. The PDE4B2 cDNA was inserted into the expression plasmids PET-32a, pPIC9K and pYeDP60, respectively, resulting in the recombinant plasmids PET-32a/PDE , pPIC9K/PDE and pYeDP60/PDE. The recombinant plasmid were transformed into E. coli strain BL21trxB(DE3), P. pastoris GS115 and WHT respectively. The transformants were induced to obtain recombinant protein. The recombinant protein was purified by His-tag affinity chromatography and identified as the recombinant human PDE4B2 by Western blot analysis. Bio-activity assay in vitro demonstrated that the recombinant protein expressed in E. coli close to the native protein.