University of Illinois at Chicago
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Mutagenesis Of Beta-Barrel Membrane Proteins To Evaluate The Role Of Sequence And Structural Motifs

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posted on 2012-12-07, 00:00 authored by Volga Pasupuleti
Proteins are a major component of any living cell, playing an important role in nutrient uptake, metabolism, transport and many other functions. The structure of the protein affects the way it functions and with the advances in NMR and X-ray crystallography extensive research is being done on hundreds of proteins on the structural level. Membrane proteins are coded by 25% of the genes in an organism’s genome and act as channels, pumps, sensors and receptors. In spite of having such vital functions, membrane proteins have been left out of the crystallographer’s repertoire probably due to the difficulty in crystallizing them in the critical environmental requirements in which they function. Previous studies suggest a series of models on the structural prediction of the ß-barrel transmembrane proteins. In those models they were able to identify sequence and spatial patterns such as alanine- tyrosine motifs, antimotifs, chaperone binding motifs, aromatic rescue of glycine and amino acid regional propensities for specific locations that are statistically important but biological significance has not been determined. To elucidate which amino acids or patterns of amino acids are important for in vivo folding and which influence thermodynamic stability of ß-barrel membrane proteins they proposed a series of experiments with outer membrane protein F (OmpF). The ompF gene was deleted from MG1655 E.coli strain and was replaced with a kanamycin resistant gene using homologous gene recombination. Site directed mutagenesis was done on pBAD24F plasmid to produce different mutants of ompF gene. Proteins expressed by wild type and mutant plasmids in the omp8 strain (ompF null) were checked on SDS-PAGE for expression and thermal stability.



Liang, Jie



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University of Illinois at Chicago

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  • Masters

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  • en

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