Cell‐free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens

Abstract

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.

Escherichia coli‐based cell free production of flagellin and ordered flagellin display on virus‐like particles

“…The open nature of the cell‐free system allows facile modification of the reaction environment, and the lack of intact cells provides for simpler purification procedures. The cell‐free platform has been shown to be useful for the production of a variety of different proteins and protein assembles including: lymphokines (Yang et al, 2005), integral membrane proteins (Wuu and Swartz, 2008), virus‐like particles (Bundy et al, 2008), Gaussia luciferase (Welsh et al, 2009), human transcription factors (Yang et al, 2009), and influenza hemagglutinin head domain antigens (Welsh et al, 2012).…”

Functional properties of flagellin as a stimulator of innate immunity

“…Currently, flagellin is mainly produced by in vivo recombinant DNA technology, and most evaluations of flagellin as an immune stimulator have used one of the two forms of flagellin from Salmonella typhimurium , FliC and FljB 6 7 8 . CFPS technology is emerging as a powerful platform for the synthesis of pharmaceutical proteins 9 10 11 12 and can produce proteins from either PCR products or plasmid templates in a few hours. The open nature of the CFPS system allows facile modification of the reaction environment, and the absence of a cell wall enables simpler purification procedures 13 .…”

The Evolution of Cell Free Biomanufacturing

“…Protein synthesis has also been improved using CFME approaches; Calhoun and Swartz [95], for example, performed chromsosomal deletions in the source cells to address the problem of cell free amino acid degradation. Gene overexpression approaches have also been used to improve protein yields [96,97]. CFME has also been used to address certain bottlenecks in CFPS such as the need for energy and cofactor regeneration in cell extracts.…”