PMID: 9533626Apr 9, 1998Paper

Oligopeptidase B: cloning and probing stability under nonequilibrium conditions

Proteins
L Polgár, F Felföldi

Abstract

Oligopeptidase B is a member of a new serine peptidase family, unrelated to the trypsin and subtilisin families. It is a potential processing enzyme of prokaryotes, being very specific for the basic amino acid pairs of polypeptides. An understanding of the kinetics of the enzyme requires the examination of its conformational stability under a variety of conditions. To this end, the enzyme was cloned from Escherichia coli HB101 by the PCR method, expressed with high yield in E. coli XL1-Blue, and purified essentially in two chromatographic steps. The denatured enzyme failed to refold, which precluded the calculation of free energy of stability, deltaG0. Therefore, the unfolding rates were measured to probe the stability against urea, pH, and heat. Denaturation processes were monitored by intrinsic fluorescence, circular dichroism, and activity measurements. A static method, intrinsic fluorescence vs. pH, was indicative of significant changes in the tertiary structure of the enzyme pH < 6 and pH > 8.5. The more sensitive dynamic methods, unfolding rates in urea and inactivation rates at high temperature, revealed increased flexibility in the protein structure between pH 6 and pH 7, where the static method did not show significant...Continue Reading

Citations

May 25, 2002·Journal of Bacteriology·Rory E MortyNorma W Andrews
Mar 7, 2006·Protein Expression and Purification·Jian-Bin YanXue-Yuan Jiang

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