Protein folding intermediates of invasin protein IbeA from Escherichia coli

Characterizing protein folding intermediates.

To determine the pathway by which a protein folds up, it is necessary to characterize the structures of folding intermediates and also to place these intermediates in the correct order on the kinetic pathway of folding. Noncovalent folding reactions are fast: typically they occur in seconds or less for small single-domain proteins. On the other hand, hours are required to obtain detailed struct...

Advances and challenges in recombinant protein expression in Escherichia coli

Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli

Background: The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. Methods: An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested ...

Is protein folding hierarchic? II. Folding intermediates and transition states.

The folding reactions of some small proteins show clear evidence of a hierarchic process, whereas others, lacking detectable intermediates, do not. Evidence from folding intermediates and transition states suggests that folding begins locally, and that the formation of native secondary structure precedes the formation of tertiary interactions, not the reverse. Some notable examples in the liter...

Hyperproduction of araC protein from Escherichia coli.

Hypersynthesis of araC protein from Escherichia coli has been accomplished. The araC gene was cloned on plasmid pBR322, and some of the noncoding DNA preceding the araC gene was removed by exonuclease digestion. Finally, a DNA fragment containing the lac promoter and ribosome binding site was placed in front the araC gene. By these means the level of araC protein was increased about 5000-fold a...