Recombinant expression and purification of cysteine-rich Granulin-7 from E.coli
Dylan T. Tran1, Anukool A. Bhopatkar2, and Vijayaraghavan Rangachari2
1Mississippi INBRE Research Scholar, Mississippi Gulf Coast Community, Gulfport, MS
2Department of Chemistry and Biochemistry, School of Mathematics and Natural Sciences, The University of Southern Mississippi, Hattiesburg, MS
Granulins (GRNs 1-7) are cysteine-rich, ~ 6 kDa repeat domains proteins that are generated by the cleavage of the precursor, progranulin (PGRN). These proteins possess multiple biological roles within normal physiology such as neurotrophic factors, immunomodulators, growth regulators etc. Pathologically, they have been implicated in neurodegenerative diseases such as Alzheimer disease (AD), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Despite these links there is a dearth of information on the structure-function relation of the individuals GRNs. The goal of this study is to successfully standardize the recombinant expression and purification protocol for one of the GRNs; GRN-7 within E.coli to allow a complete biophysical and biochemical characterization. In our lab, we have previously established a purification protocol for GRNs-3 and 5 based on immobilized metal-affinity chromatography. For GRN-7, however, we observed the presence of proteolytic cleavage products along with the purified protein. To overcome the issue of proteolytic digestion by endogenous proteases, we changed the expression system from Origami 2 to BL21 E. coli cells. The results of this project have been presented here and discussed.