Related Experiment Video
Updated: Oct 17, 2025
Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
Expression and purification of a cleavable recombinant fortilin from Escherichia coli for structure activity studies
Maranda S Cantrell1, Jackson D Wall2, Xinzhu Pu3
1Biomolecular Sciences Ph.D. Program, Boise State University, Boise, ID, 83725, USA; Department of Chemistry and Biochemistry, Boise State University, Boise, ID, 83725, USA.
Insights
Researchers developed a method to produce pure fortilin, a novel drug target for atherosclerosis beyond cholesterol reduction. This advancement enables structure-based drug discovery for cardiovascular disease, offering new therapeutic avenues.
Area of Science:
- Biochemistry
- Cardiovascular Research
- Drug Discovery
Background:
- Atherosclerosis causes significant mortality, with statins targeting LDL-cholesterol.
- Many patients have normal cholesterol, indicating other disease mechanisms.
- Human fortilin, an anti-apoptotic protein, protects macrophages, contributing to atherosclerosis.
Purpose of the Study:
- To develop a method for producing pure, bioactive recombinant fortilin.
- To enable structure-based drug discovery for atherosclerosis targeting fortilin.
- To identify optimal expression and purification conditions for fortilin.
Main Methods:
- Engineered expression constructs with various affinity tags (MBP, 6His, GST) and protease cleavage sites (TEV, HRV 3C).
- Expressed and purified fusion proteins in Escherichia coli.
- Assessed protease cleavage efficiency with varying glycine/serine (GGS) linker lengths.
Main Results:
- High expression of soluble fusion proteins was achieved.
- Fusion constructs were resistant to cleavage without linkers.
- Inclusion of at least one GGS linker enabled successful protease cleavage.
- Produced pure fortilin with conserved calcium-binding ability, confirmed by NMR.
Conclusions:
- A robust method for producing pure, functional fortilin was established.
- This method facilitates structure-activity relationship studies for fortilin.
- Fortilin represents a promising, non-cholesterol-centric drug target for atherosclerosis.
Abstract:
Complications related to atherosclerosis account for approximately 1 in 4 deaths in the United States and treatment has focused on lowering serum LDL-cholesterol levels with statins. However, approximately 50% of those diagnosed with atherosclerosis have blood cholesterol levels within normal parameters. Human fortilin is an anti-apoptotic protein and a factor in macrophage-mediated atherosclerosis and is hypothesized to protect inflammatory macrophages from apoptosis, leading to subsequent cardiac pathogenesis. Fortilin is unique because it provides a novel drug target for atherosclerosis that goes beyond lowering cholesterol and utilization of a solution nuclear magnetic resonance (NMR) spectroscopy, structure-based drug discovery approach requires milligram quantities of pure, bioactive, recombinant fortilin. Here, we designed expression constructs with different affinity tags and protease cleavage sites to find optimal conditions to obtain the quantity and purity of protein necessary for structure activity relationship studies. Plasmids encoding fortilin with maltose binding protein (MBP), 6-histidine (6His) and glutathione-S-transferase (GST), N- terminal affinity tags were expressed and purified from Escherichia coli (E. coli). Cleavage sites with tobacco etch virus (TEV) protease and human rhinovirus (HRV) 3C protease were assessed. Despite high levels of expression of soluble protein, the fusion constructs were resistant to proteinases without the inclusion of amino acids between the cleavage site and N-terminus. We surveyed constructs with increasing lengths of glycine/serine (GGS) linkers between the cleavage site and fortilin and found that inclusion of at least one GGS insert led to successful protease cleavage and pure fortilin with conserved binding to calcium as measured by NMR.
