A novel protein-engineering approach for enhancing the crystallization of recalcitrant proteins
Open Access
- Author:
- Hostetler, Zachary M
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Song Tan, Thesis Supervisor
David Scott Gilmour, Thesis Honors Advisor
Dr. Wendy Hanna-Rose, Faculty Reader - Keywords:
- structural biology
x-ray crystallography
protein
crystallization
recombinant
fusion
GFP
SUMO
hRCC1 - Abstract:
- The three-dimensional structure of a protein is critical information for a biologist because the structure of a protein can provide new insight into the mechanism of its function. One of the primary methods for determining the structure of proteins at an atomic resolution is X-ray crystallography. In practice, X-ray crystallography is limited by our ability to grow large, ordered protein crystals. Arguably, the most important determinant for protein crystallization is the protein itself. Protein engineering approaches have been employed in the past to overcome the recalcitrance of particular proteins to crystallization. For example, Suzuki and colleagues solved the structure of small polypeptides by fusing green fluorescence protein (GFP) to their N-terminal ends. I explore here the utility of GFP and small ubiquitin-related modifier (SUMO) as crystallization fusion tags for proteins. Modular DNA expression vectors that allow fusion of GFP or SUMO to a gene of interest were constructed, and fusion proteins were overexpressed in E. coli. SUMO fusion proteins demonstrated higher levels of expression and solubility as compared to their wild type or GFP-fusion counterparts. A catalytically inactive SUMO protease mutant was engineered as a cocrystallization partner to stabilize the flexible linker of the SUMO fusion proteins. Complexes of inactive SUMO protease and SUMO fusion proteins were reconstituted and isolated using size exclusion chromatography. However, preliminary crystallization trials of the SUMO-hRCC1/SUMO protease complex were unsuccessful. Future work with this crystallization system will include characterizing the binding affinity of the inactive SUMO protease, crystallizing different SUMO fusion proteins, and testing the effect of the flexible linker length on crystallization.