cDNA-directed expression of a functional zebrafish CYP1A in yeast
cDNA-directed expression of a functional zebrafish CYP1A in yeast
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DOI：
10.1016/j.aquatox.2004.07.007
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发表时间：
2004-11-18
影响因子：
4.5
通讯作者：
Buhler, DR
中科院分区：
文献类型：
--
作者：
Chung, WG;Sen, A;Buhler, DR
从斑马鱼(Danio Rerio)文库中克隆了细胞色素P4501AcDNA.2580bp的克隆(GenBank登录号：AF210727)含有一个62个碱基的5‘非翻译区，1557个碱基的编码区和962个碱基的3’非编码区。推导出的519个氨基酸残基(计算相对分子质量58,556，等电点为7.58)与虹鱼CYP1a的同源性为74%，与小鼠和人的同源性分别为57%和54%。将斑马鱼CYP1A蛋白编码区克隆到pDONR201载体上，并转化酵母表达载体pYES-DEST52。用半乳糖诱导斑马鱼CyP1a在酿酒酵母转化子中的表达，最高可达493pmolCyP1a/mg微粒体蛋白，约为8nmoL/L。在酵母中表达的重组蛋白主要以变性的P420形式存在，但当缓冲液中的氧气浓度通过脱气降低，酵母细胞保持在10℃以下时，其完整性得以保持，并在448 nm处表现出特征的还原CO差光谱最大值。重组斑马鱼CYP1A具有7-乙氧基间苯二酚O-脱乙基酶(EROD)活性，在30℃时的表观K-m(Km(App))和V-max分别为0.31+/-0.04um和0.70+/-0.10nmoL/min/nmolCYP。该重组蛋白还能代谢苯并(A)芘，其Km(App)和V-max分别为5.34+/-0.58um和1.16+/-0.13nmoL/min/nmolCYP。这些结果表明，功能斑马鱼CYP在酵母中得到了重组表达，酵母被证实为斑马鱼CyP1a异源表达的宿主，并有可能成为其他斑马鱼CyP的宿主。(C)2004爱思唯尔B.V.保留所有权利。
A cytochrome P450 1A (CYP1A) cDNA was isolated from an adult zebrafish (Danio rerio) library. The 2580-bp clone (GenBank Accession No. AF210727) contained a 62-bp 5'-unstranslated region (UTR), 1557-bp coding region and 962-bp 3'-UTR. The deduced 519-residue protein (calculated molecular weight 58,556, pI = 7.58) shared 74% identity with rainbow trout CYP1A and 57 and 54% identities with mouse and human CYP1A1s, respectively. The zebrafish CYP1A protein coding region was cloned into the pDONR201 entry vector and then transferred to a yeast expression vector pYES-DEST52. Expression of zebrafish CYP1A in Saccharomyces cerevisiae transformants was induced by galactose to a maximum level of 493 pmol CYP1A per mg microsomal protein or about 8 nmol/l of culture. Recombinant CYP1A protein expressed in yeast was mainly in the denatured P420 form under normal microsomal preparation conditions but when the oxygen concentration was reduced in the buffer by degassing and the yeast cells were maintained at less than 10 degreesC, the integrity of the CYP1A was preserved and it exhibited a characteristic reduced CO-difference spectrum maximum at 448 nm. The recombinant zebrafish CYP1A demonstrated 7-ethoxyresorufin O-deethylase (EROD) activity with an apparent K-m (Km(app)) and V-max values at 30 degreesC of 0.31 +/- 0.04 muM and 0.70 +/- 0.10 nmol/min/nmol CYP, respectively. The recombinant protein also metabolized benzo(a)pyrene with a Km(app) and V-max values of 5.34 +/- 0.58 muM and 1.16 +/- 0.13 nmol/min/nmol CYP, respectively. These results show the recombinant expression of a functional zebrafish CYP in yeast and validated yeast as a host for heterologous expression of zebrafish CYP1A and potentially for other zebrafish CYPs. (C) 2004 Elsevier B.V. All rights reserved.