Cost-effective Molecular Weight Standards for Protein Size Exclusion Chromatography
Restricted (Penn State Only)
- Author:
- Sokol, Joshua
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Song Tan, Thesis Supervisor
Ying Gu, Thesis Honors Advisor - Keywords:
- Size exclusion chromatography
Gel filtration chromatography
Globular
Liquid chromatography
Protein
Recombinant DNA
SEC-MALS
Analytical Ultracentrifugation
Sedimentation equilibrium
Metal affinity chromatography
Ion-exchange chromatography
Molecular weight - Abstract:
- Size exclusion chromatography (SEC) is useful for protein molecular weight determination when calibrated with a set of protein standards. SEC separates molecules by molecular size so an ideal standard is perfectly globular. Available standards are nonglobular and costly. The following research was performed to generate cost-effective improved SEC standards. Our criteria included: (1) the proteins should be as globular as possible, (2) the proteins should have molecular weights in round numbers from 10 kDa to 1000 kDa, (3) the proteins should be produced in large quantities using recombinant expression in Escherichia coli and standard purification techniques, and (4) the proteins should behave well in SEC, size exclusion chromatography with multi-angle light scattering (SEC-MALS), and analytical ultracentrifugation (AUC). We produced proteins that match specific, round-number molecular weights, including 10 kDa, 25 kDa, 50 kDa, 100 kDa, 250 kDa, 500 kDa, 1000 kDa, and 3000 kDa. These proteins can generate an accurate size exclusion calibration curve to calculate the molecular weight of a broad-size range of macromolecules run on SEC. Customized genes were amplified and subcloned into our T7 promotor expression system plasmids. Expression conditions were optimized by varying temperature and induction time. Hexahistidine N-terminal tags were incorporated in the proteins for purification by Talon metal affinity chromatography. Urea concentrations during Talon purification were optimized to increase recovery by reducing multivalent interactions between protein and resin. The proteins were further purified by ion-exchange chromatography and then analyzed by SEC, SEC-MALS, and AUC. We desire for this work to aid in protein characterization work reliant on molecular weight determinations using SEC, particularly for under-funded research groups.