Davis K M, Brigstock D R, Johnson P R, Crissman-Combs M A, McCarthy D W, Downing M T, Besner G E
Molecular, Cellular, and Developmental Biology Program, The Ohio State University and Children's Hospital, Columbus, Ohio, 43205, USA.
Protein Expr Purif. 1996 Aug;8(1):57-67. doi: 10.1006/prep.1996.0074.
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 C4 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.
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。由于尚未描述能够详细分析HB-EGF糖基化功能意义的重组表达系统，因此通过携带HB-EGF cDNA的中间质粒载体与痘苗病毒基因组的同源重组，构建了一种用于表达HB-EGF的重组痘苗病毒，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，通过对HeLa细胞条件培养基的放射受体和DNA合成分析表明，表达的蛋白是一种分泌型生物活性蛋白。使用肝素亲和快速蛋白质液相色谱，随后进行C4反相高效液相色谱（RP-HPLC），从条件培养基中纯化重组蛋白。RP-HPLC纯化产物的SDS-PAGE和蛋白质印迹显示，约22 kDa的免疫反应性HB-EGF蛋白经O-聚糖酶处理后降低为14 kDa蛋白。氨基酸测序揭示了一个具有天然糖基化HB-EGF特征的N末端。有趣的是，一个假定的O-连接糖基化位点的苏氨酸残基未能解析出来。该系统为评估糖基化在HB-EGF功能中的作用以及解决其他有关HB-EGF结构-功能关系的问题提供了一种有价值的方法。
