Guo Wenjin, Cleveland Brad, Davenport Thaddeus M, Lee Kelly K, Hu Shiu-Lok
Department of Pharmaceutics, University of Washington, Seattle, WA 98195, USA.
Protein Expr Purif. 2013 Jul;90(1):34-9. doi: 10.1016/j.pep.2013.04.009. Epub 2013 May 9.
Vaccinia virus (VV) has been used to express a variety of heterologous proteins, including HIV envelope (Env) glycoproteins. The Env protein is synthesized as a precursor molecule, gp160, which is cleaved into the surface antigen gp120 and the transmembrane protein gp41. Even though production of gp160 by the VV expression system has been described, its use for gp120 production is not well documented. Here we report a new procedure for the purification of gp120 from serum-containing culture supernatant of VV-infected cells. The gp120 protein was enriched to a purity better than 60% on a snowdrop (Galanthus nivalis) lectin affinity column in the presence of 0.25% zwitterionic detergent Empigen BB. After additional DEAE anion exchange and Superdex size exclusion chromatography steps, the gp120 monomer was purified free of contamination as determined by SDS-PAGE. The retention of structural integrity was confirmed by determining the affinity constant of purified gp120s to soluble CD4 and a monoclonal antibody IgG1b12, using surface plasmon resonance analysis. The purification procedure is robust and reproducible, and may find general use for glycoprotein purifications from sources where the presence of serum is desirable.
痘苗病毒（VV）已被用于表达多种异源蛋白，包括HIV包膜（Env）糖蛋白。Env蛋白以前体分子gp160的形式合成，该前体分子可裂解为表面抗原gp120和跨膜蛋白gp41。尽管已描述了通过VV表达系统生产gp160，但关于其用于生产gp120的报道并不充分。在此，我们报告了一种从VV感染细胞的含血清培养上清中纯化gp120的新方法。在0.25%两性离子去污剂Empigen BB存在的情况下，gp120蛋白在雪花莲（Galanthus nivalis）凝集素亲和柱上富集至纯度超过60%。经过额外的DEAE阴离子交换和Superdex尺寸排阻色谱步骤后，通过SDS-PAGE测定，纯化得到的gp120单体无污染物。通过表面等离子体共振分析确定纯化的gp120与可溶性CD4和单克隆抗体IgG1b12的亲和常数，证实了其结构完整性得以保留。该纯化方法稳健且可重复，可能在需要血清存在的来源中用于糖蛋白纯化方面具有广泛用途。
