A robust protocol for refolding TolC and other outer membrane components of tripartite efflux pumps from inclusion bodies.

Antibiotic resistance is a growing threat in the modern world. In Gram-negative bacteria, one factor contributing to antibiotic resistance is the tripartite efflux pumps which push antibiotics out of the cell against their concentration gradient. These pumps consist of three main protein complexes: an outer membrane protein, an inner membrane protein, and the periplasmic adapter protein, which connects the two membrane proteins. Multiple efflux pumps in resistant strains use the same outer membrane protein, TolC. This protein is a homotrimeric transmembrane membrane beta barrel with a periplasmic homotrimeric alpha-helical barrel. Until recently, isolation of folded TolC from the outer membrane was quite difficult, leading to low yields. Our lab developed an inclusion body isolation and refolding protocol to increase the yield of trimeric TolC. We identified two crucial factors that support TolC refolding: detergent choice and protein concentration and found that this method is also successful for some TolC homologs (V. cholerae VceC and C. jejuni CmeC). This chapter seeks to provide an in-depth guide for investigators wanting to refold TolC or its homologs, by giving insight into common pitfalls and other issues we have noted in our work.