Recombinant expression screening of P. aeruginosa bacterial inner membrane proteins.
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.
Department of Biological Sciences, University of Illinois at Chicago, 60607, USA.
Vidya
V
Madhavan
Forum
F
Bhatt
Constance J
CJ
Jeffery
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020539:Sequence Analysis, Protein
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins