Production of glycosylated heparin-binding EGF-like growth factor in HeLa cells using vaccinia virus
Production of glycosylated heparin-binding EGF-like growth factor in HeLa cells using vaccinia virus
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DOI：
10.1006/prep.1996.0074
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发表时间：
1996-08-01
影响因子：
1.6
通讯作者：
Besner, GE
中科院分区：
文献类型：
--
作者：
Davis, KM;Brigstock, DR;Besner, GE
肝素结合表皮生长因子样生长因子（HB-EGF）是一种22 kDa的O-糖基化蛋白质。由于允许详细分析HB-EGF糖基化的功能意义的重组表达系统尚未被描述，通过携带HB-EGF cDNA的中间质粒载体和牛痘病毒基因组的同源重组产生设计用于表达HB-EGF的重组牛痘病毒，并用于感染HeLa细胞。通过免疫荧光和放射免疫沉淀分析证实了免疫反应性HB-EGF的产生。此外，表达的蛋白质被证明是分泌的，生物活性蛋白的HeLa细胞条件培养基的放射受体和DNA合成测定。使用肝素亲和快速蛋白液相色谱法，然后用C-4反相高效液相色谱法（RP-HPLC）从条件培养基中纯化重组蛋白。RP-HPLC纯化产物的SDS-PAGE和Western印迹显示约22 kDa的免疫反应性HB-EGF蛋白，其通过用O-聚糖酶处理而降低至14 kDa蛋白。氨基酸测序显示N-末端是天然的，糖基化的HB-EGF的特征。有趣的是，一个Thr残基，这是一个假定的O-连接的糖基化位点未能得到解决。该系统提供了一种有价值的方法，用于评估糖基化在HB-EGF功能中的作用，以及解决有关HB-EGF结构-功能关系的其他问题。(C)出版社：Academic Press，Inc.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a 22-kDa, O-glycosylated protein. Because recombinant expression systems permitting a detailed analysis of the functional significance of HB-EGF glycosylation have not been described, a recombinant vaccinia virus designed to express HB-EGF was generated by homologous recombination of an intermediate plasmid vector carrying the HB-EGF cDNA and the genome of vaccinia virus and was used to infect HeLa cells. production of immunoreactive HB-EGF was confirmed by immunofluorescence and radioimmunoprecipitation analysis. Furthermore, the expressed protein was shown to be a secreted, biologically active protein by radioreceptor and DNA synthesis assays of HeLa cell conditioned medium. The recombinant protein was purified from the conditioned medium using heparin-affinity fast protein liquid chromatography followed by C-4 reverse-phase high-performance liquid chromatography (RP-HPLC). SDS-PAGE and Western blotting of the RP-HPLC-purified product showed an immunoreactive HB-EGF protein of approximately 22 kDa that was decreased to a 14-kDa protein by treatment with O-glycanase. Amino acid sequencing revealed an N-terminus that was characteristic of native, glycosylated HB-EGF. Interestingly, a Thr residue that is a putative site of O-linked glycosylation failed to be resolved. This system provides a valuable method for evaluating the role of glycosylation in HB-EGF function(s) as well as addressing other questions concerning HB-EGF structure-function relationships. (C) 1996 Academic Press, Inc.