Tavernier J, Marmenout A, Bauden R, Hauquier G, Van Ostade X, Fiers W
Laboratorium Molekulaire Biologie, Ghent, Belgium.
J Mol Biol. 1990 Jan 20;211(2):493-501. doi: 10.1016/0022-2836(90)90367-U.
To analyse the structure-function relationship of tumour necrosis factor (TNF), a set of in-frame chimeric genes was constructed by coupling appropriate segments of the human and mouse TNF coding regions. Under control of the bacteriophage lambda inducible PL promoter high level expression of these chimeric genes was obtained in Escherichia coli. Although both human and mouse TNF were produced in E. coli as soluble proteins, a reduction of solubility was observed in some of the chimeric proteins. The specific activity was variable, but in some constructs comparable to human TNF, indicating that the structural conformation of these chimeric proteins resembled the human TNF structure. Neutralization analysis using two monoclonal antibodies directed against human TNF, indicated that the regions involved in the binding of these antibodies are distributed over multiple segments of the polypeptide. Further analysis by site-directed mutagenesis of one subregion allowed the identification of the Arg131 residue as involved in the binding of both neutralizing monoclonal antibodies; an Arg131----Gln replacement abolished antibody binding but did not affect the specific activity of TNF.
为了分析肿瘤坏死因子（TNF）的结构-功能关系，通过连接人类和小鼠TNF编码区的适当片段构建了一组读框内嵌合基因。在噬菌体λ诱导型PL启动子的控制下，这些嵌合基因在大肠杆菌中获得了高水平表达。尽管人类和小鼠TNF在大肠杆菌中均作为可溶性蛋白产生，但在一些嵌合蛋白中观察到溶解性降低。比活性各不相同，但在某些构建体中与人类TNF相当，这表明这些嵌合蛋白的结构构象类似于人类TNF结构。使用两种针对人类TNF的单克隆抗体进行的中和分析表明，参与这些抗体结合的区域分布在多肽的多个片段上。通过对一个亚区域进行定点诱变的进一步分析，确定了Arg131残基参与两种中和单克隆抗体的结合；将Arg131替换为Gln消除了抗体结合，但不影响TNF的比活性。
