Tonouchi N, Oouchi N, Kashima N, Kawai M, Nagase K, Okano A, Matsui H, Yamada K, Hirano T, Kishimoto T
Central Research Laboratories, Ajinomoto Co., Inc., Kanagawa.
J Biochem. 1988 Jul;104(1):30-4. doi: 10.1093/oxfordjournals.jbchem.a122416.
BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation. Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator. In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed. A "fused" expression system was therefore developed to prepare the recombinant protein. In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide. In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred. As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P. From 1 liter of E. coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells.
BSF-2（B细胞刺激因子-2/白细胞介素-6）是淋巴因子家族的一员，负责B细胞分化。使用色氨酸启动子/操纵子和色氨酸A终止子构建了人BSF-2 cDNA的表达质粒。在含有“直接”表达质粒pBSF-2D的大肠杆菌HB101提取物中，检测到了BSF-2的活性，但未观察到过量表达。因此，开发了一种“融合”表达系统来制备重组蛋白。在该系统中，cDNA表达为人白细胞介素-2 N端肽的融合蛋白。在融合的BSF-2表达质粒pBSF-2F的情况下，观察到了包涵体，并且该蛋白发生了过量表达。由于这种融合蛋白在人白细胞介素-2和BSF-2的连接处具有苯丙氨酸-精氨酸-丙氨酸序列，可以通过用激肽释放酶和氨肽酶P处理从融合的BSF-2中加工出成熟的BSF-2。从1升大肠杆菌培养物中纯化出45毫克成熟的BSF-2；其相对生物学活性与从T细胞中纯化的天然BSF-2相当。
