Departments of Chemical Engineering and Bioengineering, Stanford University, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2014 Jan 7;111(1):125-30. doi: 10.1073/pnas.1308701110. Epub 2013 Dec 16.
The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.
2009 年大流行性 H1N1 流感病毒的迅速传播强调了需要通用流感疫苗，以广泛预防多种突变株。最近的研究重点是高度保守的血凝素（HA）茎域，该区域必须发生重大构象变化才能有效感染病毒。尽管已经证明生产多聚体细胞外结构域蛋白的分离结构域具有一定难度，但我们报告了一种使用基于大肠杆菌的无细胞蛋白合成和简单复性方案，从流感病毒 A/加利福尼亚/05/2009（H1N1）快速生产正确折叠的 HA 茎域蛋白的方法。HA 茎域蛋白 C 末端的 T4 噬菌体纤维蛋白折叠结构域诱导三聚体形成。强调新暴露的蛋白质表面，突变了几个疏水性残基，删除了两个多肽片段，并将每个单体中的二硫键数量从四个减少到两个。高 pH 值和 Brij 35 去污剂是提高复性产率最有益的因素。为了稳定 HA 茎-折叠融合物的三聚体，最后在折叠结构域单体之间和茎域单体之间引入了新的分子间二硫键。通过使用 CR6261、C179 和 FI6 抗体证实了稳定的 HA 茎域三聚体的正确免疫原性构象，这些抗体通过与 HA 茎域三聚体结合来阻断流感感染。这些结果表明广泛保护性疫苗具有很大的前景，并且还展示了一种生产复杂多聚体蛋白的单个结构域的独特方法。
