Membrane proteins play essential roles in cellular functions, but their investigation presents unique challenges. Understanding their structure and function is crucial, as they are significant drug targets, with over 50% of approved drugs targeting membrane proteins.

Our service is based on our expertise in membrane protein production, including case studies on ion channels and G protein-coupled receptors (GPCRs). By employing state-of-the-art technologies and methodologies, we facilitate the efficient production of high-quality, recombinant membrane proteins, facilitating the drug development process for our collaborators.

Production of membrane proteins

Overexpression

Various expression systems

  • Mammalian cells
  • Insect cells (BEVS)
  • E. coli (for protein fragments)

Construct design

  • Type and position of the affinity tag
  • Tag separation via linker/protease cleavage site
  • Truncations/mutations

Different strategies of protein overexpression

  • Infection/transduction with recombinant baculovirus
  • Transient transfection

Optimization of the expression conditions

  • Cell lines, media, MOI, IVP/L, time of expression, temperature, additives

Membrane solubilization and protein stabilization

Various methods of cell lysis

  • Homogenization
  • Sonication
  • Freeze-thaw

Methods of membrane solubilization

  • Direct solubilization during cell lysis
  • Solubilization of the purified/isolated membranes

Screening of solubilization parameters

  • Type of detergent
  • Cholesterol-based additives
  • Mixture/concentration of detergents
  • Solubilization time
  • Buffer composition

Purification 

A broad range of chromatographic methods

  • Affinity chromatography (His-tag, GST, MBP, Strep, Twin-Strep, FLAG, c-Myc)
  • Size-exclusion chromatography, resolution range (1kDa –5 MDa)
  • Ion exchange chromatography

Removal of the tag during the purification process

  • Choice of different proteases

Characterization

Identity

  • MS (peptide mapping) or N-terminal sequencing

Purity

  • Densitometric analysis of CBB-stained gel

Overall fold and stability

  • nanoDSF

Homogeneity and oligomerization status (if possible)

  • HPLC-SEC-MALS