Membrane Protein Technology
Specialized production of GPCRs, ion channels, transporters, and complex membrane receptors. Integrating eukaryotic expression (Sf9, Expi293F), membrane isolation, detergent solubilization screening, FPLC purification, and MSP nanodisc reconstitution.
How Membrane Proteins Are Made
Membrane proteins require expression hosts that support membrane insertion, proper folding, and essential post-translational processing. Recombinant genes are introduced into Sf9 insect cells or Expi293F mammalian cells. After expression, cellular membranes are isolated and the target protein is gently extracted into a compatible detergent solution before chromatographic purification.
Why Membrane Proteins Require Specialized Workflows
Unlike soluble proteins, membrane proteins depend on a hydrophobic lipid bilayer environment to maintain their native three-dimensional conformation. Once removed from the membrane, they are prone to misfolding and irreversible aggregation unless shielded in optimized detergent micelles or reconstituted into native-like lipid nanodiscs.
Specialized 9-Stage Pipeline
Stages of Membrane Protein Production
From sequence design through membrane fractionation, detergent extraction, and nanodisc reconstitution.
Target evaluation and construct design
Evaluate transmembrane topology, domain boundaries, loop regions, and glycosylation sites. Incorporate affinity tags, protease cleavage sites, and stabilizing mutations when appropriate.
Host selection & expression screening
Test construct variants in Sf9 and Expi293F systems. Evaluate expression yield, membrane localization, and initial solubility on a small scale before volume scale-up.
Scale-up and expression culture
Propagate high-density cultures under optimized conditions to maximize functional target yield in the host membrane fraction.
Membrane harvesting & preparation
Lyse cells gently and isolate enriched crude membrane fractions by differential ultracentrifugation, removing soluble cytoplasmic contaminants.
Detergent extraction and screening
Screen detergent panels (DDM, LMNG, GDN, CHAPS, digitonin) with and without cholesteryl hemisuccinate (CHS) to identify conditions that solubilize the target without causing denaturation.
Clarification and initial capture
High-speed centrifugation removes unsolubilized debris. The detergent-protein micelle is captured via immobilized metal affinity (IMAC), FLAG, or Strep-tag chromatography.
Chromatographic polishing
FPLC size-exclusion chromatography (SEC) and ion-exchange under detergent maintenance resolve monomeric/oligomeric states from aggregate species.
Membrane-mimetic stabilization
Maintain the purified protein in optimized detergent micelles, or reconstitute into proteoliposomes or MSP-based lipid nanodiscs for detergent-free stability.
Characterization and functional QC
Evaluate purity (SDS-PAGE), oligomeric homogeneity (analytical SEC/DLS), thermal stability (nanoDSF/CPM), and ligand binding competence (SPR/BLI) in the final format.
Stabilization and Delivery Formats
The final format is tailored to downstream applications: cryo-EM structure determination, surface plasmon resonance (SPR), antibody screening, or biochemical assays.
Detergent Micelles
Purified proteins are kept soluble in optimized non-denaturing detergents (e.g. DDM, LMNG, GDN) with CHS. Ideal for rapid functional testing and crystallography.
Proteoliposomes
Reconstitution into synthetic or native lipid vesicles. Re-establishes a continuous lipid bilayer for transport assays, ion flux studies, and patch-clamp electrophysiology.
MSP Nanodiscs
Membrane Scaffold Protein (MSP) wraps around a discoidal lipid bilayer patch containing a single membrane protein. Detergent-free, soluble, and monodisperse for Cryo-EM and SPR.
Membrane Protein Quality and Functional Assessment
Because membrane proteins can lose conformation without showing degradation on a denaturing gel, functional integrity is verified using binding assays, thermal denaturation profiles, and monodispersity analysis in the chosen delivery format.