Recombinant expression, purification, crystallization and preliminary X‐ray diffraction analysis of Haemophilus influenzae BamD and BamCD complex. Issue 2 (1st February 2015)

• Record Type: Journal Article
• Authors: Lei, Jintang; Cai, Xun; Ma, Xiaodan; Zhang, Li; Li, Yuwen; Dong, Xue; St Geme, Joseph; Meng, Guoyu
• Is Part Of: Acta crystallographica. Volume 71:Issue 2(2015:Feb.)
• Page Start: 234
• Page End: 238
• Publication Date: 2015-02-01
• Subjects: Crystallography -- Periodicals; Crystals -- Periodicals; 548
• DOI: 10.1107/S2053230X14027319
• Languages: English
• ISSNs: 2053-230X

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>The Bam machinery, which is highly conserved from bacteria to humans, is well recognized as the apparatus responsible for the insertion and folding of most outer membrane proteins in Gram‐negative bacteria. In <italic>Escherichia coli</italic>, the Bam machinery consists of five components (<italic>i.e.</italic> BamA, BamB, BamC, BamD and BamE). In comparison, there are only four partners in <italic>Haemophilus influenzae</italic>: a BamB homologue is not found in its genome. In this study, the recombinant expression, purification, crystallization and preliminary X‐ray diffraction analysis of <italic>H. influenzae</italic> BamD and BamCD complex are reported. The genes encoding BamC and BamD were cloned into a pET vector and expressed in <italic>E. coli</italic>. Affinity, ion‐exchange and gel‐filtration chromatography were used to obtain high‐purity protein for further crystallographic characterization. Using the hanging‐drop vapour‐diffusion technique, BamD and BamCD protein crystals of suitable size were obtained using protein concentrations of 70 and 50 mg ml<sup>−1</sup>, respectively. Preliminary X‐ray diffraction analysis showed that the BamD crystals diffracted to 4.0 Å resolution and belonged to space group <italic>P</italic>2<sub>1</sub>2<sub>1</sub>2<sub>1</sub>, with unit‐cell parameters <italic>a</italic> = 54.5, <italic>b</italic> = 130.5, <italic>c</italic> = 154.7 Å. The BamCD crystals diffracted to 3.8 Å resolution and belonged to space group <italic>I</italic>2<sub>1</sub>2<sub>1</sub>2<sub>1</sub>, with unit‐cell parameters <italic>a</italic> = 101.6, <italic>b</italic> = 114.1, <italic>c</italic> = 234.9 Å.</p> </abstract>