The expression, purification, and structure determination of Bama from E. Coli

Abstract

In gram-negative bacteria, assembly of outer membrane proteins requires the multicomponent β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved integral outer membrane protein. To understand how BamA facilitates outer membrane protein biogenesis, it is important to obtain suffi cient amounts of purifi ed recombinant BamA protein for in vitro functional analysis and structure determination. In this chapter, we describe the protocol that we used in our laboratory for the cloning, expression, and purifi cation of E. coli BamA and its N-terminal deletion variants for in vitro functional studies and for structure determination of the β-barrel domain alone (residues 426-810).