Screening, Purification and Concentration of Membrane Proteins

Screening, purification and concentration of membrane proteins

Membrane proteins (MPs) are important targets for pharmaceutical products and for the design of novel sensors, synthetic membranes and bioelectronics devices. The hydrophobic exterior of MPs necessitates their extraction and stabilization in detergent micelles. Low-yield MPs are typically concentrated by ultrafiltration (UF) in commercially available centrifugal filers which is accompanied by concomitant increase in concentration of detergent micelles. Excessive detergent can interfere with stability and characterization of MPs. Here, we discuss two methods of obtaining high concentration of MPs without increasing detergent concentration using light-driven sodium and chloride transporters, KR2 and halorhodopsin (pHR), as model proteins.  From an earlier study, we know that concentration polarization (CP) during UF promotes detergent passage while retaining MPs. Thus to promote CP, we designed a flat-bottomed centrifugal filter unit that retrofits into commercially available falcon tubes and tested its performance using four commonly employed non-ionic MP detergents, octyl-beta-D maltoside (OM), decyl-beta-D maltoside (DM), dodecyl-beta-D maltoside (DDM) and octylÐbeta-D glucoside (OG). Detergent transmission was significantly greater for our new flat-bottomed filter compared to currently available modules due to the higher degree of CP. Alternately, increasing the extraction of MPs from lipid bilayers using longer alkyl chain detergents and higher salt concentration is another means of improving yield. We attribute this improvement in extraction to the increase in micellar hydrophobicity with chain length and salt concentrations; this was verified using StokeÕs shift of the solvatochromic chromophore, DSSN+. However, while longer chain detergents show better extraction, they also form larger micelles that are difficult to remove by UF. Thus, there exists a trade-off between choosing a detergent that will enable efficient extraction of MP while showing easier removal during subsequent ultrafiltration. To screen and optimize aqueous detergent and salt/ buffer conditions for histidine-tagged MP purification, we used nickel-functionalized membranes in a 96 well plate format. In addition to MP downstream processing, our findings can be extended to other detergentÐmediated separation/extraction processes.

 

 

 

 

 

 

 

Figure. A. Detergents play an important role in membrane protein (MP) solubilization, purification and post-purification processing, i.e., concentration for subsequent characterization assays. Detergents with increasing chain length promotes MP extraction but also lead to simultaneous concentration of detergent during ultrafiltration. B. Ultrafiltration of different detergent and membrane protein-detergent systems show higher transmission of detergent in flat (designed) versus commercial centrifugal filters.