Hammacher A, Reid G E, Moritz R L, Simpson R J
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research/Walter and Eliza Hall Institute of Medical Research, Victoria, Australia.
Biomed Chromatogr. 1997 Nov-Dec;11(6):337-42. doi: 10.1002/(SICI)1099-0801(199711)11:6<337::AID-BMC687>3.0.CO;2-E.
We have identified a preparation of recombinant murine interleukin-6 (mIL-6) that, in addition to the anticipated product, also contained approximately equal amounts of mIL-6 with a C-terminal pentapeptide extension. The extension mutant was generated by readthrough of the stopcodon, and termination at a second in-frame stopcodon 12 base pairs 3' in the expression vector. Aliquots of the preparation were subjected to proteolytic digestion with Asp-N and Lys-C-endopeptidase. The resultant peptides were separated by reversed-phase capillary HPLC, and analysed using a combination of mass spectrometry and N-terminal sequence analysis. These data revealed a C-terminal pentapeptide (Gln-Gly-Ser-Val-Asp) extension, with the authentic stopcodon being translated as glutamine. The extension mutant was isolated by reversed-phase HPLC and shown to have similar mitogenic activity to mIL-6 on murine hybridoma 7TD1 cells.
我们鉴定出一种重组鼠白细胞介素-6（mIL-6）制剂，该制剂除了预期产物外，还含有大致等量的带有C端五肽延伸的mIL-6。延伸突变体是通过终止密码子通读产生的，并在表达载体中3'端12个碱基对处的第二个框内终止密码子处终止。将该制剂的等分试样用天冬氨酸-N和赖氨酸-C内肽酶进行蛋白水解消化。所得肽通过反相毛细管HPLC分离，并结合质谱和N端序列分析进行分析。这些数据揭示了一个C端五肽（谷氨酰胺-甘氨酸-丝氨酸-缬氨酸-天冬氨酸）延伸，真实的终止密码子被翻译为谷氨酰胺。通过反相HPLC分离出延伸突变体，并显示其对鼠杂交瘤7TD1细胞具有与mIL-6相似的促有丝分裂活性。
