Optimization of Size Exclusion Chromatography Protein Standards
Restricted (Penn State Only)
- Author:
- Mongia, Advay
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Song Tan, Thesis Supervisor
Joseph C. Reese, Thesis Honors Advisor - Keywords:
- Size Exclusion Chromatography
Protein Standards
protein expression
protein purification
gel filtration chromatography
bacterioferritin - Abstract:
- Size exclusion chromatography, also known as gel filtration chromatography, is a widely used purification technique separating molecules based upon their size. The protein standards used for this method are crucial to estimate protein sizes; however, commercially sold standards are often expensive. In conjunction with other undergraduate students in the lab, we aim to develop a new standard that will be easily made and cost effective. Previous work in the lab has included similar projects that have successfully created new, cost-effective DNA and protein ladders for gel electrophoresis. The proteins used in the gel filtration standard will cover a wide range of molecular weight, from 10 to 3,000 kilodaltons. The focus of this work is the protein Mycobacterium tuberculosis bacterioferritin B (HISBfrB) as a candidate at 500 kilodaltons. The first steps entailed inserting the gene into our T7 promoter expression vector and transforming it into competent E. coli cells. The proteins were expressed in BL21(DE3)pLysS cells, and conditions such as temperature and time were manipulated such that high expression was displayed. High yield after purification was then obtained using Talon purification. Optimization of purification was conducted with the addition of urea to decrease any self-aggregation of the protein. HISBfrB has been expressed at 18℃ and purified in the presence of 3M urea at the small-scale level. The broader goal of the project will be to simplify protein analysis by gel filtration chromatography and allow the scientific community to employ the standard in their work.