Luk S K, Jay E, Jay F T
Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada.
J Biol Chem. 1990 Aug 5;265(22):13314-9.
The structure-function relationships for the human interferon gamma (HuIFN-gamma) were studied using recombinant variants that had various deletions at the carboxyl terminus. Four COOH-terminal deletion variants were constructed that contained the amino-terminal 122, 117, 111, and 106 amino acid residues. These variants were constructed by specific DNA modifications and were expressed in Escherichia coli. The deletion of 21 amino acid residues resulted in only 2- and 3-fold reduction in the antiviral and antiproliferative specific activities, respectively. Thus, the carboxyl-terminal 21 residues are not directly involved in the function of the HuIFN-gamma. The level of intracellular accumulation was also decreased by 3-5-fold. Further deletions of 26, 32, and 37 residues from the COOH terminus resulted in the lack of detectable activity as well as in 50-100-fold reduction in the level of accumulation in the bacterial cell. However, each of the modified plasmids was found to have comparable efficiency in directing the production of the respective variant molecules relative to the full-length HuIFN-gamma molecule in an in vitro transcription-translation assay. Thus, the failure of some of the deletion variant molecules to accumulate in the E. coli cell is likely due to their instability in vitro. The loss of the COOH-terminal 21 amino acid residues of HuIFN-gamma also resulted in a substantial reduction in the ability of the molecule to be renatured in vitro from the treatment with chaotrophic agents, a method frequently used to extract and purify recombinant polypeptides from E. coli host. The latter result may account for some earlier reports which inferred the involvement of the COOH terminus in the functions of the HuIFN-gamma molecule due to their failure to detect activity upon deletions of only 11-18 residues.
利用在羧基末端有不同缺失的重组变体研究了人γ干扰素（HuIFN-γ）的结构-功能关系。构建了四个羧基末端缺失变体，它们分别包含氨基末端的122、117、111和106个氨基酸残基。这些变体通过特定的DNA修饰构建而成，并在大肠杆菌中表达。缺失21个氨基酸残基分别导致抗病毒和抗增殖比活性仅降低2倍和3倍。因此，羧基末端的21个残基不直接参与HuIFN-γ的功能。细胞内积累水平也降低了3至5倍。从羧基末端进一步缺失26、32和37个残基导致无法检测到活性，同时细菌细胞中的积累水平降低了50至100倍。然而，在体外转录-翻译试验中，发现每个修饰质粒在指导各自变体分子产生方面相对于全长HuIFN-γ分子具有相当的效率。因此，一些缺失变体分子未能在大肠杆菌细胞中积累可能是由于它们在体外不稳定。HuIFN-γ羧基末端21个氨基酸残基的缺失也导致该分子在用离液剂处理后体外复性能力大幅降低，离液剂是从大肠杆菌宿主中提取和纯化重组多肽常用的方法。后一结果可能解释了一些早期报告，这些报告由于仅缺失11至18个残基后未检测到活性而推断羧基末端参与了HuIFN-γ分子的功能。
