(301e) Using Ultrafiltration to Concentrate Detergent-Solubilized Membrane Proteins without Concentrating Detergents

Authors: 
Feroz, H., Pennsylvania State University
Vandervelden, C., University of Kansas
Ikwuagwu, B., University of Pittsburgh
Ferlez, B., Pennsylvania State University
Baker, C. S., Pennsylvania State University
Lugar, D. J., Pennsylvania State University
Grzelakowski, M., Applied Biomimetic Inc.
Golbeck, J. H., Pennsylvania State University
Zydney, A. L., The Pennsylvania State University
Kumar, M., Pennsylvania State University

Membrane
proteins (MPs) carry out various physiological functions in living organisms
and as such are important targets for pharmaceutical products and for the
design of novel sensors and synthetic membranes (Kim et al. 2012; Kowalczyk et al. ; Shen et al. 2014). The challenge with using MPs for such applications
lies in purifying high concentrations of functional MPs. The hydrophobic
exterior of MPs necessitates their stabilization in detergent micelles. Due to
low yields obtained, MPs often need to be concentrated for subsequent
biophysical characterization or use in applications. The widely-used ultrafiltration-based
concentration of MPs in commercial filters often leads to a concomitant increase
in concentration of empty detergent micelles. The resulting high detergent
concentrations can destabilize MPs, interfere with subsequent structural and
biophysical characterization of MPs, and present challenges in applications. We
present a detailed analysis of the hydrodynamic conditions that selectively
promote detergent passage during MP ultrafiltration. We studied two different
classes of transporter MPs, the bacterial water channel protein, Aquaporin Z
(AqpZ) and the archaeal light-driven chloride pump, Halorhodopsin (pHR) in nonionic
detergents, octyl–β-D glucoside (OG) and decyl-β-D maltoside (DM) respectively.
The observed sieving coefficient (So)- the ratio of detergent concentration
in permeate to that in retentate, was evaluated in the stirred cell and
centrifugal systems. The sieving coefficient for
detergent for
both pure detergent and MP-detergent systems was much greater in the stirred
cell than in the commercial filters (Figure 1A). Thus, almost complete
transmission of detergent can be achieved by operating under conditions that encourage
concentration polarization (CP). This contradicts current approaches in soluble
protein concentration systems, including centrifugal filters, which are designed
to maximize flux by minimizing CP (Figure 1B). Thus a rethinking of the design
of ultrafiltration-based systems of MP concentration to promote CP may be
required for downstream processing of MPs.

References

Kim Y-R,
Jung S, Ryu H, Yoo Y-E, Kim SM, Jeon T-J. 2012. Synthetic biomimetic membranes
and their sensor applications. Sensors 12(7):9530-9550.

Kowalczyk
SW, Blosser TR, Dekker C. Biomimetic nanopores: learning from and about nature.
Trends in Biotechnology 29(12):607-614.

Shen Y, Saboe
PO, Sines IT, Erbakan M, Kumar M. 2014. Biomimetic membranes: a review. Journal
of Membrane Science 454:359-381.