| Title |
Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions
|
|---|---|
| Published in |
European Biophysics Journal, July 2018
|
| DOI | 10.1007/s00249-018-1325-z |
| Pubmed ID | |
| Authors |
Aalishaa A. Azam, Jean M. Kinder, G. Nasir Khan, Ade Alase, Pikyee Ma, Yang Liu, James R. Ault, Peter J. F. Henderson, Babur Z. Chowdhry, Bruce D. Alexander, Stephen E. Harding, Mary K. Phillips-Jones |
| Abstract |
Despite the importance of membrane proteins in cellular processes, studies of these hydrophobic proteins present major technical challenges, including expression and purification for structural and biophysical studies. A modified strategy of that proposed previously by Saidijam et al. (2005) and others, for the routine expression of bacterial membrane proteins involved in environmental sensing and antimicrobial resistance (AMR), is proposed which results in purification of sufficient proteins for biophysical experiments. We report expression successes amongst a collection of enterococcal vancomycin resistance membrane proteins: VanTG, VanTG-M transporter domain, VanZ and the previously characterised VanS (A-type) histidine protein kinase (HPK). Using the same strategy, we report on the successful amplification and purification of intact BlpH and ComD2 HPKs of Streptococcus pneumoniae. Near-UV circular dichroism revealed both recombinant proteins bound their pheromone ligands BlpC and CSP2. Interestingly, CSP1 also interacted with ComD. Finally, we evaluate the alternative strategy for studying sensory HPKs involving isolated soluble sensory domain fragments, exemplified by successful production of VicKESD of Enterococcus faecalis VicK. Purified VicKESD possessed secondary structure post-purification. Thermal denaturation experiments using far-UV CD, a technique which can be revealing regarding ligand binding, revealed that: (a) VicKESD denaturation occurs between 15 and 50 °C; and (b) reducing conditions did not detectably affect denaturation profiles suggesting reducing conditions per se are not directly sensed by VicKESD. Our findings provide information on a modified strategy for the successful expression, production and/or storage of bacterial membrane HPKs, AMR proteins and sensory domains for their future crystallisation, and ligand binding studies. |
X Demographics
The data shown below were collected from the profiles of 2 X users who shared this research output. Click here to find out more about how the information was compiled.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United Kingdom | 1 | 50% |
| United States | 1 | 50% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 2 | 100% |
Mendeley readers
The data shown below were compiled from readership statistics for 20 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 20 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 5 | 25% |
| Student > Master | 3 | 15% |
| Student > Doctoral Student | 2 | 10% |
| Lecturer > Senior Lecturer | 2 | 10% |
| Student > Ph. D. Student | 2 | 10% |
| Other | 3 | 15% |
| Unknown | 3 | 15% |
| Readers by discipline | Count | As % |
|---|---|---|
| Agricultural and Biological Sciences | 5 | 25% |
| Chemistry | 4 | 20% |
| Biochemistry, Genetics and Molecular Biology | 4 | 20% |
| Veterinary Science and Veterinary Medicine | 1 | 5% |
| Medicine and Dentistry | 1 | 5% |
| Other | 1 | 5% |
| Unknown | 4 | 20% |
Attention Score in Context
This research output has an Altmetric Attention Score of 2. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 19 June 2020.
All research outputs
#15,015,838
of 23,098,660 outputs
Outputs from European Biophysics Journal
#304
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Outputs of similar age
#197,961
of 329,833 outputs
Outputs of similar age from European Biophysics Journal
#3
of 9 outputs
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