Improving extraction, purification and characterization of membrane proteins

Open Access
Author:
Oh, Hyeonji
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Manish Kumar, Thesis Supervisor
  • Enrique Daniel Gomez, Honors Advisor
Keywords:
  • membrane protein
  • purification
  • characterization
  • light-driven ion pump
Abstract:
Membrane proteins (MPs) have gained steady increase in interest due to its critical importance in biomedical research. In current extraction processes, MPs are solubilized and stabilized by specialized non-ionic detergents to stabilize their hydrophobic exteriors. However, these free detergent molecules and empty detergent micelles (not containing MPs) are often retained together with the extracted MPs in subsequent ultrafiltration (UF) steps which are required to obtain higher concentration of MPs needed for characterization process and eventual use. This poses a challenge as the retained detergent may destabilize MPs and interfere with their functional/biophysical characterization. To address this challenge, the effect of detergent selection on extraction and ultrafiltration was studied in this work. Also, a flat-bottomed centrifugal filter was designed to concentrate MPs and remove free detergents molecules/empty detergent micelles with higher efficiencies. Two biomedically-relevant MPs, halorhodopsin (pHR) and KR2 were used as models for complex 7 transmembrane helix containing G-coupled Protein Receptors (GPCRs). Common employed MP detergents were used to investigate the influence of detergent selection and to test the performance of this filter over a wide range of salt concentrations. Detergent with longer alkyl chains such as dodecyl maltoside (DDM) which has a 12-carbon alkyl chain showed more efficient extraction than the ones with shorter alkyl chains due to its higher hydrophobicity, larger micelle size, and lower CMC. Detergent passage of flat-bottomed centrifugal filter was significantly higher compared to the commercial centrifugal filters due to its higher concentration polarization (CP).