Evaluation of a Low Cost, Downstream Purification Process for Griffithsin - a Potential Broad Spectrum Viral Entry Inhibitor Produced in Engineered E. coli

Abstract

<p>HIV infections remain a major public health issue with no current cure: in 2015, the disease led to the deaths of 1.1 million people [1]. A viable preventative is Griffithsin (GRFT), a lectin that binds and neutralizes the HIV viral envelope by blocking glycoproteins that are needed for the virus to recognize target cells. The protein is being studied as a highly effective pre-exposure prophylactic therapy [2]. However for it to become a preventative, particularly in the developing world, product cost and stability are key barriers that need to be resolved before widespread use. We have developed an initial and low cost downstream process that requires three steps to purify GRFT from E. coli fermentations taking advantage of the protein’s unique melting temperature, isoelectric point, and size. SDS-PAGE revealed that the purification steps designed are capable of retaining GRFT while LS/MS/MS proteomics has proven enrichment of GRFT throughout the process. This study also successfully establishes a baseline of what proteins act as contaminants identifying 22 top gene deletion candidates guiding future strain engineering efforts.</p>