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Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

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PDF 1472-6750-10-83-s1.pdf (99KB) Table listing the 87 Pseudomonas aeruginosa target proteins in the study, including the ORF numbers, gene names, protein names, predicted number of transmembrane helices and percent of amino acids in transmembrane helices, the calculated GRAVY score, and the results of the expression studies at several temperatures in two E. coli strains. PDF
Title: Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins
Author(s): Madhavan, Vidya; Bhatt, Forum; Jeffery, Constance J.
Subject(s): recombinant transmembrane proteins
Abstract: BACKGROUND: Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. RESULTS: Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. CONCLUSIONS: In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this one could aid in overcoming the technical bottlenecks in working with TM proteins and could potentially aid in increasing the rate of structure determination.
Issue Date: 2010-11-29
Publisher: BioMed Central
Citation Info: Madhavan, V., Bhatt, F., & Jeffery, C. J. 2010. Recombinant expression screening of P. aeruginosa bacterial inner membrane proteins. BMC Biotechnology,10: 83. DOI: 10.1186/1472-6750-10-83
Type: Article
Description: © 2010 Madhavan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The original version is available through BioMed Central at DOI: 10.1186/1472-6750-10-83.
URI: http://hdl.handle.net/10027/7782
ISSN: 1472-6750
Sponsor: This project was supported by grants to CJJ from the National Science Foundation and the Society for Biomolecular Sciences.
Date Available in INDIGO: 2011-05-27
 

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