Cell-free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens
Article first published online: 16 AUG 2012
Copyright © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 109, Issue 12, pages 2962–2969, December 2012
How to Cite
Welsh, J. P., Lu, Y., He, X.-S., Greenberg, H. B. and Swartz, J. R. (2012), Cell-free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens. Biotechnol. Bioeng., 109: 2962–2969. doi: 10.1002/bit.24581
- Issue published online: 25 OCT 2012
- Article first published online: 16 AUG 2012
- Accepted manuscript online: 21 JUN 2012 08:51AM EST
- Manuscript Accepted: 13 JUN 2012
- Manuscript Revised: 10 JUN 2012
- Manuscript Received: 2 MAY 2012
- NIH. Grant Numbers: AI057229, AI090019
- cell-free protein synthesis
In order to effectively combat pandemic influenza threats, there is a need for more rapid and robust vaccine production methods. In this article, we demonstrate E. coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce domains from the protein hemagglutinin (HA), which is present on the surface of the influenza virus. The portion of the HA coding sequence for the “head” domain from the 2009 pandemic H1N1 strain was first optimized for E. coli expression. The protein domain was then produced in CFPS reactions and purified in soluble form first as a monomer and then as a trimer by a C-terminal addition of the T4 bacteriophage foldon domain. Production of soluble trimeric HA head domain was enhanced by introducing stabilizing amino acid mutations to the construct in order to avoid aggregation. Trimerization was verified using size exclusion HPLC, and the stabilized HA head domain trimer was more effectively recognized by antibodies from pandemic H1N1 influenza vaccine recipients than was the monomer and also bound to sialic acids more strongly, indicating that the trimers are correctly formed and could be potentially effective as vaccines. Biotechnol. Bioeng. 2012; 109: 2962–2969. © 2012 Wiley Periodicals, Inc.