Adolf G R, Frühbeis B, Hauptmann R, Kalsner I, Maurer-Fogy I, Ostermann E, Patzelt E, Schwendenwein R, Sommergruber W, Zöphel A
Department of Cell Biology, Ernst Boehringer--Institut für Arzneimittelforschung, Bender + Co Ges mbH, Vienna, Austria.
Biochim Biophys Acta. 1991 Jun 13;1089(2):167-74. doi: 10.1016/0167-4781(91)90004-6.
A gene encoding human interferon omega-1 (IFN-omega 1) was isolated from a cosmid library, sequenced and expressed in Chinese hamster ovary (CHO) cells under the control of an SV40-derived promoter/enhancer sequence. Culture supernatants of stably transfected cell clones contained biologically active IFN-omega 1 at concentrations up to 10 micrograms/l. Amplification of the expression vector containing a dhfr gene under methotrexate selection pressure resulted in yields up to 200 micrograms/l. Production of IFN-omega 1 was further enhanced 2- to 3-fold by propagation of the cells in the presence of n-butyrate. IFN-omega 1 was purified from culture supernatants by monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase HPLC and size-exclusion HPLC. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two bands of about the same intensity with apparent molecular masses of 24.5 and 22.5 kDa. Upon treatment with peptide:N-glycosidase F, both bands were shifted to lower molecular masses (20.5 and 18.5 kDa), indicating that CHO cell-derived IFN-omega 1 is glycosylated; Asn-78 was identified as the glycosylation site. Analysis of the carbohydrate moiety using glycosidases and lectins revealed the presence of biantennary complex oligosaccharides containing neuraminic acid. Amino acid sequencing showed that only about 40% of the molecules have the expected N-terminus, whereas the others carry two additional amino acids derived from the signal sequence. C-terminal amino acid sequencing using carboxypeptidase P demonstrated that the smaller form of the protein lacks nine amino acids. Disulfide bridges were shown to connect Cys residues 1 and 99 as well as 29 and 139, respectively, as in IFN-alpha. The specific antiviral activity of recombinant, glycosylated human IFN-omega 1 on human cells was 2.6 x 10(8) IU/mg, not significantly different from that of the authentic, human leukocyte-derived protein.
从黏粒文库中分离出编码人ω-1干扰素（IFN-ω1）的基因，进行测序，并在SV40衍生的启动子/增强子序列控制下在中国仓鼠卵巢（CHO）细胞中表达。稳定转染细胞克隆的培养上清液中含有浓度高达10微克/升的生物活性IFN-ω1。在甲氨蝶呤选择压力下对含有二氢叶酸还原酶（dhfr）基因的表达载体进行扩增，产量可达200微克/升。在丁酸钠存在下培养细胞，IFN-ω1的产量进一步提高了2至3倍。通过单克隆抗体亲和层析从培养上清液中纯化IFN-ω1。通过反相高效液相色谱（HPLC）和尺寸排阻HPLC测定，所得蛋白质的纯度至少为95%。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）显示两条强度大致相同的条带，表观分子量分别为24.5和22.5 kDa。用肽：N-糖苷酶F处理后，两条带均迁移至较低分子量（20.5和18.5 kDa），表明CHO细胞来源的IFN-ω1是糖基化的；天冬酰胺-78被确定为糖基化位点。使用糖苷酶和凝集素对碳水化合物部分进行分析，发现存在含有神经氨酸的双天线复杂寡糖。氨基酸测序表明，只有约40%的分子具有预期的N端，而其他分子则携带两个源自信号序列的额外氨基酸。使用羧肽酶P进行C端氨基酸测序表明，较小形式的蛋白质缺少九个氨基酸。如在IFN-α中一样，二硫键分别连接半胱氨酸残基1和99以及29和139。重组糖基化人IFN-ω1对人细胞的特异性抗病毒活性为2.6×10⁸国际单位/毫克，与天然的人白细胞来源蛋白无显著差异。
