Cell-free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens.
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.
Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, California 94305-5025, USA.
John P
JP
Welsh
Yuan
Y
Lu
Xiao-Song
XS
He
Harry B
HB
Greenberg
James R
JR
Swartz
D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000235:genetics; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D060748:Fetuins / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007252:Influenza Vaccines / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000956:Antigens, Viral; D060748:Fetuins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D011994:Recombinant Proteins; D019158:N-Acetylneuraminic Acid