Isolation, characterization, and expression in Escherichia coli of a cDNA encoding rat heme oxygenase-2.
Isolation, characterization, and expression in Escherichia coli of a cDNA encoding rat heme oxygenase-2.
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DOI：
10.1016/s0021-9258(19)39141-0
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发表时间：
1990-05
期刊：
影响因子：
--
通讯作者：
Mitch O. Rotenberg;M. Maines
中科院分区：
文献类型：
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作者：
Mitch O. Rotenberg;M. Maines
在最近的一项研究中（Cruse，I.，和Maines，M.D.（1988）J.Biol.Chem.263，3348-3353），我们报道了通过免疫学筛选λ gt 11中的大鼠睾丸cDNA文库，分离编码血红素加氧酶-2的一部分的小cDNA片段。我们现在已经使用这个274个碱基对（bp）的cDNA片段作为杂交探针，用于重新筛选相同的文库，从而回收了一些额外的阳性分离株。其中，分别约900、1100和1300 bp的三个候选物随后被亚克隆和测序。尽管长度不同，但发现这些克隆的序列在其他方面相同。此外，分离物18 B的长度为1284 bp，与通过大鼠睾丸总RNA和poly（A）+RNA的北方印迹杂交分析检测到的单个mRNA种类（约1300-1350个核苷酸）的长度完全一致。该全长或近全长cDNA编码315个氨基酸的蛋白质，分子量为35，757，与血红素加氧酶-2的估计分子量36，000非常一致。当在大肠杆菌中表达时，cDNA编码与血红素加氧酶-2抗血清交叉反应的蛋白质（如通过Western免疫印迹分析），并在细菌可溶性细胞提取物中产生高水平的血红素加氧酶活性。最后，对血红素加氧酶-2cDNA序列的计算机分析表明，血红素加氧酶-2蛋白的预测氨基酸序列和亲水性特征与血红素加氧酶-1表现出相似性。
In a recent study (Cruse, I., and Maines, M.D. (1988) J. Biol. Chem. 263, 3348-3353), we reported the isolation of a small cDNA fragment encoding a portion of heme oxygenase-2 through immunological screening of a rat testis cDNA library in lambda gt11. We have now used this 274-base pair (bp) cDNA fragment as a hybridization probe for rescreening of the same library, and have thereby recovered a number of additional positive isolates. Of these, three candidates of approximately 900, 1100, and 1300 bp, respectively, were subsequently subcloned and sequenced. Although differing in length, the sequences of these clones were found to be otherwise identical. Moreover, the length of isolate 18B, 1284 bp, corresponded well with that of the single mRNA species (approximately 1300-1350 nucleotides) detected through Northern blot hybridization analysis of rat testis total and poly(A)+RNA. This full- or near full-length cDNA encodes a 315-amino acid protein with a molecular weight of 35,757, in good agreement with the 36,000 estimated molecular weight of heme oxygenase-2. When expressed in Escherichia coli, cDNA encodes a protein that cross-reacts with heme oxygenase-2 antiserum (as assayed by Western immunoblotting) and yields high levels of heme oxygenase activity in bacterial soluble cell extracts. Finally, computer analysis of the heme oxygenase-2 cDNA sequence indicates that the predicted amino acid sequence and hydropathy profile of the heme oxygenase-2 protein exhibit similarity with heme oxygenase-1.