Recombinant human cytidine deaminase: Expression, purification, and characterization
Recombinant human cytidine deaminase: Expression, purification, and characterization
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DOI：
10.1006/prep.1996.0097
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发表时间：
1996-09-01
影响因子：
1.6
通讯作者：
Vita, A
中科院分区：
文献类型：
--
作者：
Vincenzetti, S;Cambi, A;Vita, A
利用两种特异性引物对人胞苷脱氨酶（CDA）进行逆转录酶PCR筛选，获得了编码CDA的互补DNA （cDNA）。将cDNA片段连接到表达载体pTrc99-A中，经异丙基硫代β - d -半乳糖苷（IPTG）诱导，在大肠杆菌中表达。cDNA的核苷酸序列与Laliberte和Momparler （Cancer Res. 54, 5401-5407, 1994）发表的序列一致。它包含一个438 bp的开放阅读框，编码146个氨基酸的多肽，预测分子质量为16.2 kDa。在大肠杆菌中表达的蛋白具有较高的胞苷脱氨酶活性，经凝胶过滤和sds -聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定其分子量分别为57 kDa和16 kDa。这两个值与已经发表的结果一致，表明人类CDA包含三到四个相同的亚基。交联实验表明该酶为四聚体。重组CDA通过热失活和亲和层析的快速纯化达到均匀性。最终酶制剂的比活性为105 U/mg，相对于萃取物纯化约88倍，通过SDS-PAGE判断其纯度为bb0 98%。电感耦合等离子体发射光谱（ICP-OES）分析表明，每个亚基中存在1个Zn原子。由于CDA可引起几种抗肿瘤胞苷类似物的脱胺作用，因此对该重组酶进行了动力学表征，并对几种嘧啶核苷类似物作为潜在底物和抑制剂进行了测试。所获得的结果与先前报道的纯化人胎盘CDA的结果非常一致。(C) 1996学术出版社，Inc.
The complementary DNA (cDNA) coding for human cytidine deaminase (CDA) was obtained using two specific primers to screen RNA from peripheral blood polymorphonuclear leukocytes by reverse transcriptase PCR. The cDNA fragment was ligated into the expression vector pTrc99-A and expressed in Escherichia coli following induction with isopropyl-l-thio-beta-D-galactopyranoside (IPTG). The nucleotide sequence of the cDNA corresponded to that published by Laliberte and Momparler (Cancer Res. 54, 5401-5407, 1994). It contained a 438-bp open reading frame encoding a polypeptide of 146 amino acids with a predicted molecular mass of 16.2 kDa. The protein expressed in E. coli showed high cytidine deaminase activity and its molecular mass was estimated to be 57 kDa by gel filtration and 16 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Both values are in agreement with those already published and suggest that human CDA contains three or four identical subunits. Cross-linking experiment indicated that the enzyme is a tetramer. The recombinant CDA has been purified to homogeneity by a rapid procedure consisting of heat inactivation followed by affinity chromatography. The final enzyme preparation showed a specific activity of 105 U/mg, corresponding to about 88-fold purification with respect to the elude extract and was judged to be >98% pure by SDS-PAGE. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis revealed the presence of 1 atom of Zn per subunit. Since CDA causes the deamination of several antitumoral cytidine-analog drugs, the recombinant enzyme was characterized kinetically and several pyrimidine nucleoside analogs were tested as potential substrates and inhibitors. The results obtained agreed closely with those previously reported for the purified human placenta CDA. (C) 1996 Academic Press, Inc.