- Production of Membrane Proteins: Strategies for Expression and Isolation.
- Membrane Proteins;
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In addition, some integral MPs are monotopic, and are embedded in only one leaflet of the bilayer. In contrast, peripheral MPs do not span the phospholipid bilayer.
Optimizing Membrane Protein Expression
Membrane proteins and receptors are the largest category of druggable targets studied in the pharmaceutical industry. Approximately two-thirds of currently available therapeutic molecules target one or more MPs. Where available, membrane protein crystal structures facilitate elucidation of their functions and discovery of mechanisms of MP-drug molecule interactions.
Methods to characterize MPs are limited by the lack of extraction protocols and reagents that allow sufficient amounts of MPs to be obtained from various cell types without cross-contamination from other protein fractions.
There are many strategies to extract MPs from eukaryotic cell lines and tissues, including subcellular fractionation e. Most of these traditional protocols are laborious, time-consuming and require expensive ultracentrifugation equipment. The Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit enables small-scale solubilization and enrichment of integral and membrane-associated proteins from cultured cells and tissues using a simple reagent-based procedure and a bench-top microcentrifuge Figure 1.
With the earlier-generation Mem-PER Kit, extraction resulted in a viscous detergent-based hydrophobic fraction containing MPs, which required further processing before downstream procedures such as protein concentration determination, SDS-PAGE, and western blotting.
- Membrane Proteins!
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- Membrane Proteins – Production and Functional Characterization, Volume 556.
The procedure for the newly developed Mem-PER Plus Kit requires no phase-separation step; thus the extraction of MPs is simple, highly reproducible, and compatible with downstream applications. We used these kits to extract membrane proteins from a tissue sample and two strains of cultured cells see METHODS section for details. We compared overall yields of the resulting cytoplasmic and membrane protein fractions Figure 2.
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The purity low cross-contamination of membrane and cytosolic protein fractions is as important as yield. To verify that the high yields obtained with the Mem-PER Plus Kit were also high-quality extractions of membrane proteins, we performed western blot analysis of membrane and cytoplasmic fractions Figure 3 obtained from cell and tissue samples.
Probing for common membrane protein markers showed that the membrane proteins COX-IV and pan-Cadherin were highly enriched in the membrane fraction, with very low cross-contamination into the cytosolic fraction.
Jurkat cells are non-adherent and do not express cadherins, thus no cadherin was detected in that membrane fraction. Figure 3.
Designed as a research-level guide to current strategies and methods of membrane protein production on the small to intermediate scale, this practice-oriented book provides detailed, step-by-step laboratory protocols as well as an explanation of the principles behind each method, together with a discussion of its relative advantages and disadvantages. Following an introductory section on current challenges in membrane protein production, the book goes on to look at expression systems, emerging methods and approaches, and protein specific considerations.
Case studies illustrate how to select or sample the optimal production system for any desired membrane protein, saving both time and money on the laboratory as well as the technical production scale. Unique in its coverage of "difficult" proteins with large membrane-embedded domains, proteins from extremophiles, peripheral membrane proteins, and protein fragments.
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