Expression, purification and crystallization of a membrane-associated, catalytically active type I signal peptidase fromStaphylococcus aureus

Staphylococcus aureusinfections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets.S. aureushas a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, thespsBgene fromS. aureusNewman strain was cloned and overexpressed inEscherichia coli. After exploring many different protein-modification constructs, SpsB was expressed as a fusion protein with maltose-binding protein and crystallized by hanging-drop vapour diffusion. The crystals belonged to the monoclinic space groupP21and diffracted to 2.05 Å resolution. The crystal structure of SpsB is anticipated to provide structural insight into Gram-positive signal peptidases and to aid in the development of antibacterial agents that target type I signal peptidases.