(158b) 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.