Dear All,

On behalf of a colleague of mine I would like to post a question regarding membrane protein purification. Since both of us have no/limited experience with membrane protein purification (from insect cells), I hope that some of you could help us. The protein contains a C-terminal his-tag and is expressed in sf9 insect cells and is localised in the membrane. The purified protein will be inserted into liposomes to study the function of this protein regarding transport of molecules.  

What would be the best way to isolate this protein from its membrane? Testing different solubilisation methods and various detergents?

Any suggestions or protocols are highly appreciated!

Best,

Patrick

Dear Patrick,

The most important point is the detergent extraction; not always the detergent during extraction must be the final one for lipsomes. Here is some literature on the field, and of course you can give him my email if he needs more help. Affinity purification and GF are the main chromatographies he can use. IEX is a little problematic since the high concentration of the protein in the resin during the capture step. HIC is not relevant here.

Mario

Literature Membrane Proteins

Detergent selection for enhanced extraction of membrane proteins - B. Arachea et al.- 
        Protein Expression and Purification 86 (2012) 12–20  (pdf)

Expression and Purification of Membrane Proteins -J.Kubicek et al.- Methods in Enzymology, Volume 541 (2014)  (pdf)

Saccharomyces cerevisiae–Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography. - Scharff-Poulsen P, Pedersen PA (2013) - PLoS ONE 8(10): e76851. doi:10.1371/journal.pone.0076851  (pdf)

A general path for large-scale solubilization of cellular proteins: From membrane receptors to multiprotein complexes
        - F.Pullara et al.- Protein Expression and Purification 87 (2013) 111–119  (pdf) (pdf Supl)
High-throughput expression and purification of membrane proteins - F.Mancia et al.- 
        J. Struct. Biol. (2010), doi:10.1016/j.jsb.2010.03.021  (pdf)
A Fluorescence-Detection Size-Exclusion Chromatography-Based Thermostability Assay for Membrane Protein Precrystallization 
        Screening - M.Hattori et al.- Structure 20, 1293–1299, August 8, 2012  (pdf) 
An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability -
        J.Fan et al.- Protein Expr. Purif. (2011), doi:10.1016/j.pep.2011.02.010   (pdf) 
 

Dear Patrick,

I agree with the suggestions and the excellent selection of literature that Mario has posted. The only thing that I would be careful about is the statement about HIC. I agree that it is not widely used but I would not generalize and say that HIC would not be applicable to detergent-solubilized membrane proteins.

In fact, once I have applied HIC for a membrane protein that was solubilized in a charged detergent (deoxycholate), bound to phenyl-sepharose and eluted by changing the detergent in the buffer to a neutral detergent (octyl-glucoside) which changed the binding to the resin significantly. Of course, this might have been an exception, but aren't we facing exceptional proteins over and over again?

I just wanted to add this to avoid that people are discouraged to consider HIC as an option.

Best regards

Hüseyin

Thank you Mario and Hüseyin for your suggestions! I will pass the information on to my colleague.

Best,

Patrick

A very important point that I forget, is that the key in a good purification of membrane proteins is the isolation of clean membranes before detergent extraction