Overexpression and purification of avian ovomucoid third domains in Escherichia coli.
Overexpression and purification of avian ovomucoid third domains in Escherichia coli.
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禽卵类粘蛋白第三结构域在大肠杆菌中的过表达和纯化。
DOI：
10.1093/protein/6.2.221
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Markley,JL
中科院分区：
文献类型：
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作者：
Hinck,AP;Walkenhorst,WF;Westler,WM;Choe,S;Markley,JL
由于缺乏一种有效的过度生产这种蛋白质的程序，对卵粘蛋白结构域的基因工程研究一直受到阻碍。本文提出的新方案实现了鸡卵粘蛋白第三结构域(OMCHI3)的分离，分离水平为22 mg纯蛋白/1个大肠杆菌培养基组分。将OMCHI3基因与葡萄球菌核酸酶基因(SNase)的3‘端融合。嵌合基因的表达受噬菌体T7启动子强转录和翻译信号的控制。在异丙基-β-D-半乳糖苷诱导下，含有目的质粒的细胞有效地过量生产蛋白质(占总可溶性蛋白质的30%)。然后，对应于OMCHI3的56个残基片段被溴化氰(CNBr)切割，位于核酸酶-OMCHI3连接处的基因工程蛋氨酸残基(OMCHI3缺乏内部蛋氨酸)被释放。SDS-PAGE、酶抑制研究和核磁共振波谱分析均表明，重组OMCHI3具有与天然来源OMCHI3相同的性质。该表达系统适用于生产[98%U15N]OMCHI3。对表达载体OMTKY3进行了突变，使其高效表达了与OMCHI3不同的OMTKY3基因。由于许多其他禽类卵粘蛋白结构域也缺乏蛋氨酸残基，该方法应该适合于大规模生产和同位素标记具有各种抑制特异性的同源蛋白酶抑制剂。
Genetic engineering studies of ovomucoid domains have been hindered by the lack of an efficient procedure for overproducing this protein. The novel scheme presented here has led to the isolation of chicken ovomucoid third domain (OMCHI3) at a level of 22 mg pure protein/1Escherichia coliculture medium. The gene coding for OMCHI3 was fused to the 3' end of the gene encoding staphylococcal nuclease (SNase). Expression of the chimeric gene was placed under control of the strong transcription and translation signals of the phage T7 promoter. Upon isopropyl-β-D-galactopyranoside induction, the cells harboring the target plasmid efficiently overproduced the protein (30% of the total soluble protein). The 56-residue fragment corresponding to OMCHI3 was then liberated by cyanogen bromide (CNBr) cleavage at a genetically engineered methionine residue located at the nuclease-OMCHI3 junction (OMCHI3 lacks an internal methionine). SDS-PAGE, enzyme inhibition studies and NMR spectroscopy all indicated that the recombinant OMCHI3 has properties identical to those of OMCHI3 isolated from its natural source. The expression system was easily adapted for the production of [98% U15N] OMCHI3. The expression vector was mutated for overexpression of turkey ovomucoid third domain (OMTKY3), which differs from OMCHI3 by three amino acid substitutions. Since many other avian ovomucoid domains also lack methionine residues, this approach should be suitable for large-scale production and isotope labeling of homologous proteinase inhibitors with a variety of inhibitory specificities.