Expression, purification and characterization of the Biotin transporter from Staphylococcus aureus.

Abstract

ECF transporters are a group of newly defined ABC-like modular transporters and they are composed of three main elements: 1) a high-affinity membrane-embedded substrate binding protein (S component), 2) a membrane-spanning protein (T component), and 3) two identical or homologous ATPases (A, A’components) which resemble the nucleotide binding domains in ABC transporters. Staphylococcus aureus biotin transporter (SaBioMNY) belongs to the subgroup II ECF transporters which are characterized by their shared use of energy coupling module (AT module) by several S components, with each having a different substrate preference. Therefore, characterizing the S families in ECF transporters are important for us to gain new knowledge about the mechanism of subgroup II ECF transporters. Besides, laboratory has developed a series of biotin analogues with antibacterial activity against S. aureus. Previous studies have demonstrated that these compounds were capable of binding to the S component of S. aureus(i.e.SaBioY). It was reasonable to speculate that these biotin analogues were transported across the S. aureus cells by the biotin transporter BioY. To further improve the antibacterial potency and selectivity, the binding and translocation mode of these compounds across the bacterial membrane via SaBioY needs to be defined. By utilizing a filter disk diffusion assay, I determined that the susceptibility of E. coli BL21 to antibiotics (erythromycin, streptomycin and chloramphenicol) was significantly increased when wild type SaBioY was heterologously overexpressed in the cells. A library of SaBioY mutants was also screened in this assay and the overexpression of all the mutants surprisingly increased the sensitivity of E.coli cells to all three antibiotics compared to the un-induced one. One exceptional mutant was the D157K/K160E that was able to restore the tolerance of cells to the antimicrobial agents. I reasoned recombinant SaBioY adopted a functional channel in the membrane of E. coli for low molecular weight antibiotics to diffuse through. In addition, I also found that R75, D157 and K160 are essential to the surrogate transport pathway since a single amino acid change can dramatically alter the sensitivity of E. coli cells to antibiotics compared to the wild type one. To further characterize the biotin core transporter SaBioY, I attempted to purify recombinant SaBioY from E. coliBL21 (DE3). The optimized conditions for expressing SaBioY were determined to be 1) culturing cells at 25°C, 2) using the richer potassium buffered TB growth medium and 3) using a high concentration of IPTG (0.8 mM). I have also developed a system for the scalable purification of this integral membrane protein using SDS as a solubilizer. 9.7mg of SDS-solubilized SaBioY (with expected molecular weight of 19,492 Da) was obtained from 2 liters of culture after IMAC purification, with 90% purity determined by Commassie staining gel. A small panel of available mild detergents was subsequently tested for their efficiency of extracting membrane protein from natural lipids with TrionX-100 giving the best extraction efficiency. This present study paves the way for further detergent screening and purification of SaBioY.