Kabashima T, Kitazono A, Kitano A, Ito K, Yoshimoto T
School of Pharmaceutical Sciences, Nagasaki University, Nagasaki. t-kabashima @cc.nagasaki-u.ac.jp
J Biochem. 1997 Sep;122(3):601-5. doi: 10.1093/oxfordjournals.jbchem.a021795.
We cloned and sequenced the Serratia marcescens prolyl aminopeptidase (SPAP) gene. Nucleotide sequence analysis revealed an open reading frame of 951 bp, encoding a protein of 317 amino acids with a predicted molecular weight of 36,083. The expressed enzyme was purified about 90-fold on columns of Toyopearl HW65C and DEAE-Toyopearl, with an activity recovery of 30%. The apparent molecular weight of the purified enzyme was 36,000 and 38,000 as estimated by SDS-PAGE and gel filtration, respectively. The enzyme was not inhibited by diisopropyl phosphofluoridate (DFP) or phenylmethylsulfonyl fluoride (PMSF), but was markedly inhibited by 3,4-dichloroisocoumarin (DCIC). Crystals of the enzyme were grown by the hanging drop vapor diffusion method using PEG6000 as a precipitant at pH 6.5. The crystals are tetragonal with cell dimensions a= b =65.6 A, and c=169.8 A, a space group P4(1)2(1)2 or P4(3)2(1)2, and probably contain one monomer in the asymmetric unit. They diffract to at least 2.22 A resolution.
我们克隆并测序了粘质沙雷氏菌脯氨酰氨肽酶（SPAP）基因。核苷酸序列分析显示一个951 bp的开放阅读框，编码一个317个氨基酸的蛋白质，预测分子量为36,083。表达的酶在Toyopearl HW65C和DEAE-Toyopearl柱上纯化了约90倍，活性回收率为30%。通过SDS-PAGE和凝胶过滤估计，纯化酶的表观分子量分别为36,000和38,000。该酶不受二异丙基氟磷酸酯（DFP）或苯甲基磺酰氟（PMSF）抑制，但受到3,4-二氯异香豆素（DCIC）的显著抑制。使用PEG6000作为沉淀剂，在pH 6.5条件下，通过悬滴气相扩散法培养该酶的晶体。晶体为四方晶系，晶胞参数a = b = 65.6 Å，c = 169.8 Å，空间群为P4(1)2(1)2或P4(3)2(1)2，不对称单元中可能包含一个单体。它们的衍射分辨率至少为2.22 Å。
