Rocque W J, Holmes W D, Patel I R, Dougherty R W, Ittoop O, Overton L, Hoffman C R, Wisely G B, Willard D H, Luther M A
Department of Molecular Sciences, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709, USA.
Protein Expr Purif. 1997 Mar;9(2):191-202. doi: 10.1006/prep.1996.0683.
We have overexpressed in a baculovirus expression system, and purified to > 95% homogeneity, milligram quantities of a human recombinant rolipram-sensitive cAMP phosphodiesterase, HSPDE4B2B (amino acid residues 81-564). The protein expression levels were approximately 8 mg of HSPDE4B2B (81-564) per liter of Sf9 cells. The Km of the purified enzyme for cAMP was 4 microM and the Ki for the Type 4 phosphodiesterase-specific inhibitor (R)-rolipram was 0.6 microM. The specific activity of the purified protein was 40 mumol/min/mg protein. A nonequilibrium filter binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with a Kd of 1.5 nM and a stoichiometry of 0.05-0.3 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Equilibrium dialysis experiments revealed a single binding constant of 140 nM with a stoichiometry of 0.75 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Size exclusion chromatography and analytical ultracentrifugation experiments suggest that the protein exists in multiple association states larger than a monomer. Proteolysis experiments revealed a 43-kDa fragment that contained catalytic and rolipram-inhibitable activities, but the fragment showed no high-affinity (R)-rolipram binding. Based on the proteolytic cleavage studies a 43-kDa protein was constructed, expressed, and purified. This protein, HSPDE4B2B (152-528), had Km and Vmax similar to those of the HSPDE4B2B (81-564) protein, but did not exhibit high-affinity (R)-rolipram binding. The protein did show low-affinity (R)-rolipram binding using the equilibrium binding assay. These results show that a low-affinity binding site for (R)-rolipram is solely contained within the catalytic domain of HSPDE4B2B, whereas high-affinity (R)-rolipram binding requires residues within the catalytic domain and residues flanking N- and/or C-terminal to the catalytic region.
我们已在杆状病毒表达系统中进行了过表达，并将人重组罗利普兰敏感的环磷酸腺苷磷酸二酯酶HSPDE4B2B（氨基酸残基81 - 564）纯化至>95%的同质性，产量达毫克级。蛋白质表达水平约为每升Sf9细胞8毫克HSPDE4B2B（81 - 564）。纯化后的酶对环磷酸腺苷的Km为4微摩尔，对4型磷酸二酯酶特异性抑制剂（R）-罗利普兰的Ki为0.6微摩尔。纯化蛋白的比活性为40微摩尔/分钟/毫克蛋白。非平衡滤膜结合试验显示纯化后的酶上存在一个高亲和力的（R）-罗利普兰结合位点，Kd为1.5纳摩尔，化学计量比为每摩尔HSPDE4B2B（81 - 564）含0.05 - 0.3摩尔的（R）-罗利普兰。平衡透析实验显示单一结合常数为140纳摩尔，化学计量比为每摩尔HSPDE4B2B（81 - 564）含0.75摩尔的（R）-罗利普兰。尺寸排阻色谱和分析超速离心实验表明该蛋白以大于单体的多种缔合状态存在。蛋白水解实验揭示了一个43千道尔顿的片段，其具有催化活性和罗利普兰抑制活性，但该片段未显示出高亲和力的（R）-罗利普兰结合。基于蛋白水解切割研究构建、表达并纯化了一种43千道尔顿的蛋白。该蛋白HSPDE4B2B（152 - 528）的Km和Vmax与HSPDE4B2B（81 - 564）蛋白相似，但未表现出高亲和力的（R）-罗利普兰结合。使用平衡结合试验时，该蛋白确实显示出低亲和力的（R）-罗利普兰结合。这些结果表明，（R）-罗利普兰的低亲和力结合位点仅存在于HSPDE4B2B的催化结构域内，而高亲和力的（R）-罗利普兰结合则需要催化结构域内以及催化区域N端和/或C端侧翼的残基。
