title: 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 sufficient amounts of purified 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 purification 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).
authors.affiliation: National Laboratory of Biomacromolecules, National Center of Protein Science-Beijing, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Dongchun
authors.initials: D
authors.lastname: Ni
authors.affiliation: National Laboratory of Biomacromolecules, National Center of Protein Science-Beijing, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. yihuahuang@ibp.ac.cn.
authors.forename: Yihua
authors.initials: Y
authors.lastname: Huang
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000166:cytology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D002479:Inclusion Bodies / Q000378:metabolism; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D029968:Escherichia coli Proteins
keywords: BamA; Outer membrane protein; Outer membrane protein biogenesis; Protein refolding