Collection and characterisation of bacterial membrane proteins

Abstract

A general strategy for the ampli¢ed expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative K K-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His) 6 at the C-terminus, and its puri¢cation in mg quantities. The retention of structural and functional integrity was veri¢ed by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and puri¢cation is extended to additional membrane proteins from H. pylori and from other bacteria. ß

Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

“…3, lanes 3 and 4). The phenomenon of changed migration behavior on SDS-PAGE gels has been reported for other glycosyltransferases possessing transmembrane domains, e.g., WbaP (53) and WchA (27), and also for membrane proteins in general (33). This may be due to the hydrophobicity of the transmembrane domain, a high binding capacity for SDS, or the retention of secondary structure accelerating passage through the gel.…”

The Hydantoin Transport Protein from Microbacterium liquefaciens

“…6), extending the success of using plasmid pTTQ18 for the expression of various kinds of membrane transporters (16,28). The produced MhpH 6 was targeted to the inner membrane fraction, and the slight appearance in the outer membrane fraction might be caused by the contamination of outer membranes with inner membrane fractions.…”

“…For the synthesis of 3 source. An expression vector for the production of MhpH 6 protein was constructed by using the vector pTTQ18 (5,16,21). The gene hyuP encoding the Mhp protein was amplified from the chromosomal DNA of the strain by PCR using the following primers: hyuP upstream primer with EcoRI site, 5Ј-CGTCA ATGAATTCGACACCCATCGAAGAGGCT-3Ј, and hyuP downstream primer with PstI site, 5Ј-TCCTTCTCCTGCAGGGTACTGCTTCTCGGTGGG-3Ј.…”

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

“…Cloning and amplification of expression of the PucI protein in E. coli was achieved using a strategy that we and others have found successful with a wide range of bacterial and archaeal membrane proteins (Ward et al, 1999;Saidijam et al, 2003;Szakonyi et al, 2007;Ma et al, 2008Ma et al, , 2013Bettaney et al, 2013). This involved design of PCR primers (forward, 59-CCGGAATTCGCATATGAAA-TTAAAAGAGAGTCAGCAGCAATCCA-39, and reverse, 59-AAAAC-TGCAGCTTCAGCCTGGCGGACCTGCGCATGTT-39) to extract and amplify the pucI gene from B. subtilis genomic DNA, introducing EcoRI and Pst I restriction sites at the 59 and 39 ends, respectively.…”