Proteobacterial Antimicrobial Compound Efflux (PACE) Family Transport Proteins from Gram-Negative Bacteria: A Strategy for Gene Cloning, Amplified Expression and Large-Scale Purification

Proteobacterial Antimicrobial Compound Efflux (PACE) family proteins are one of seven types of multidrug efflux pumps in Gram-negative bacteria. PACE proteins can actively efflux synthetic biocides, including the antiseptic chlorhexidine, whilst their physiological substrates are polyamines. PACE proteins contain four putative transmembrane-spanning alpha-helices, and experimental evidence suggests that they mainly exist and function in a dimeric state, but the high-resolution structural organisation and molecular mechanism of PACE proteins are yet to be elucidated experimentally. As an essential first step to achieve this, we employed a strategy for gene cloning, expression screening and large-scale purification of representative PACE proteins. The genes of 24 proteins were successfully cloned into IPTG-inducible plasmid pTTQ18 directly upstream from a His6-tag coding sequence and transformed into E. coli BL21(DE3) cells. Small-scale expression tests identified seven proteins amplified at a sufficient level for larger-scale cultures and purification. Based on results from 30-litre fermentor cultures and inner membrane preparations, four proteins (A1S_2063, Fbal_3166, STY_3166, Tmarg_opt) representing distinct phylogenetic groups of the PACE family, were progressed to detergent solubilisation and purification. These proteins had purities of 86, 84, 80 and 78%, and purification yields of 1.1, 1.1, 1.3 and 1.0 mg/litre cell culture, respectively. The detergent-solubilised purified proteins had far-UV circular dichroism spectra consistent with alpha-helical secondary structure, producing melting temperatures of 46.7, 34.2, 32.6 and 37.6 C, respectively. A1S_2063 was most stable and might be best for structure elucidation. Secondary structure in all purified proteins appeared reasonably stable for performing biochemical and biophysical experiments up to 25 C.