Graduation Year
2021
Document Type
Thesis
Degree
M.S.P.H.
Degree Name
MS in Public Health (M.S.P.H.)
Degree Granting Department
Global Health
Major Professor
John Adams, Ph.D.
Committee Member
Francis Ntumngia, Ph.D.
Committee Member
Michael Teng, Ph.D.
Keywords
HEK293, Polyethylenimine, Protein Expression, Spike Protein, Transfection
Abstract
With the COVID-19 pandemic showing no signs of slowing down, large-scale antigenic protein production is still needed for surveillance using serologic assays. From screening to vaccines to biotherapeutics, being able to produce the proteins for these assays is essential; however, the current gold standard method for producing SARS-CoV-2 spike proteins is prohibitively expensive for most research groups.
Alternative methods of transfecting mammalian cells to produce recombinant proteins that are relatively inexpensive have been used for years. Unlike the expensive, commercially available lipid-based methods, other established methods such as polyethyleneimine (PEI), are considerably easier, and cheaper to meet the needs of most laboratories.This thesis research evaluated ExpiFectamine compared to PEI as methods to produce the antigenic receptor-binding domain (RBD) of SARS-CoV-2 Spike protein for standard serologic assays. The results obtained from the PEI method had consistently lower yields with different concentrations of PEI and different incubation periods, yielding 20-90% less protein than the ExpiFectamine method. In addition, the purity and functionality of the proteins in standard serologic assays were less than with the ExpiFectamine method.
Therefore, we could not conclude if the quality of the recombinant spike protein using the PEI method is adequate to serve as a substitute for ExpiFectamine product in standard serologic assays. While the significantly lower cost offers a viable alternative for many research studies, the poorer yield and purity in our studies indicate further optimization is needed to assure reliable production of high-quality recombinant protein.
Scholar Commons Citation
Ninaltowski, Nichole, "Investigation of an Alternative Protocol for the Production of SARS-CoV-2 Antigenic Proteins" (2021). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/9201
