Recombinant Expression And Comparison Of Full-length Human PDE4B2and Truncated Mutant

The full-length human cyclic nucleotide phosphodiesterase PDE4B2（hPDE4B2: Genebank accession no. M97515） gene was truncated to retainits catalytic domain （152to528aa）; the encoding sequences of thefull-length and truncated mutants were connected to the His-SUMOprokaryotic expression vector pReceiver-B13and two recombinantplasmids of His-SUMO-PDEB2were constructed and transformed into E.coli BL21（DE3）. The full-length hPDE and truncated mutants, respectively,were induced by1.0mM and0.2mM IPTG for20h at160C to obtain thefusion proteins of molecular weight were about77kDa and64kDa. One-stepNi-NTA affinity chromatography purified those two proteins.Using calf intestinal alkaline phosphatase （CIAP）coupled malachitegreen（MLG） assay of phosphate, the highest specific activity of thepurified full-length hPDE4B2was0.056U·mg^-1, while that of the truncatedmutant was1.0U·mg^-1. K_m of full-length hPDE4B2was8.5±0.1μM （n=2）, K_m of the truncated mutant was54±4μM（n=5）. Inhibition constants ofrolipram were11nM （n=2） on the full-length hPDE4while0.33μM （n=3） on the truncated mutant; Ki of papaverine was5.3μM （n=2） on thefull-length hPDE4while3.4μM （n=4） on the truncated mutant. Takentogether, the truncated mutant has higher specific actyivity, but exhibiteddifferent affinities for putative inhibitors and was thus excluded from theuse as an alternative target for screening PDE4inhibitors.The full-length fused hPDE4after purification at30ug/ml in reactionmixtures caused no significant interference with malachite green assay ofphosphate even the denaturated proteins were not removed before assay.Using microplate reader system with a proper quantity of the full-lengthhPDE4B2, its Kmwas consistent with that estimated via malachite greenassay of phosphate with a spectrophotometer; at60μM cAMP, Kiofrolipram was consistent with estimated via malachite green assay ofphosphate with a spectrophotometer. Taken together, the full-length fusedhPDE4was suitabel for high-throughput-screening of PDE4inhibitors withthe microplate reader system.