Dr. Eric R. Geertsma
Junior Professor
Biocenter N200/1.08
Phone +49-(0)69-798-29255
ax: +49-(0)69-798 29244


Beate Braungart
Administrative assistant
Biocenter N200/1.10
Phone +49-(0)69-798-29238
Fax: +49-(0)69-798 29244


Membrane protein quality control

As membrane protein overexpression often results in the production of aggregated, non-functional material, additional verification is required to determine the folding quality of the protein produced. Traditionally this was done by fractionation and determining the amount of protein present in the membrane vesicles or by monitoring the activity of the protein, provided a suitable activity assay was available. However, these techniques are laborious and thus not suitable for the routine analysis of many constructs or conditions.

The recognition of green fluorescent protein (GFP) as a folding indicator (Waldo et al. 1999, Drew et al. 2001) has enabled a very rapid alternative approach to determine the amount of folded protein during expression trials. As proper folding of GFP fused to the C-terminus of a target protein depends on the correct folding of the latter, only the well-folded fusion protein becomes fluorescent.
We observed that the GFP-fusion system could not only be used to quantify the amount of folded material, but also the misfolded fraction. This is particularly useful during expression trials: an expression condition that gives a low GFP fluorescence can result from both too low induction and too high induction. Whereas the former requires an increase in the amount of messenger RNA, the latter would actually benefit from a decreased expression rate allowing the cell more time to properly process the nascent chain. Our method is based on differential migration of folded and misfolded GFP fusion proteins during SDS-PAGE. Subsequent immunodetection of both species allows simultaneous quantification of the levels of folded and aggregated protein present.

Relevant Publication

Geertsma ER, Groeneveld M, Slotboom DJ, Poolman B. (2008)
Quality control of overexpressed membrane proteins.

Proc Natl Acad Sci U S A. 2008 Apr 15;105(15):5722-7.