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Protein Expr Purif. 2010 Aug;72(2):223-33. doi: 10.1016/j.pep.2010.03.019. Epub 2010 Mar 30.

Characteristics of an extensive Mycobacterium avium subspecies paratuberculosis recombinant protein set.

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1
National Animal Disease Center, USDA-ARS, Ames, IA 50010, United States. john.bannantine@ars.usda.gov

Abstract

In the first step of a comprehensive large-scale antigen discovery project, 651 genes of Mycobacterium avium subspecies paratuberculosis were expressed in Escherichia coli. All of these were purified by affinity chromatography, dialyzed in phosphate buffered saline, and analyzed on SDS-PAGE gels. Collectively, these purified recombinant proteins represent 14.9% of the total M. avium subsp. paratuberculosis proteome. This volume of protein expression and purification has yielded unique observations that may be missed in smaller scale expression and purification projects. For example, the 252 putative membrane proteins predicted by PSORTb analysis, resulted in lower average expression yields (3.51mg/l culture) than the 176 predicted cytoplasmic proteins (7.27mg/l culture). A few proteins (MAP0107c, MAP3169c and MAP3640) appear to promote lysis of E. coli since there was a drop in optical density of the growth culture minutes after the inducing agent was added. Certain M. avium subsp. paratuberculosis proteins, when expressed in E. coli changed the color of the column resin or appearance of harvested cell pellets. Finally, 19 proteins showed an absorbance maximum at 260nm rather than 280nm that was attributed to binding of nucleic acid during purification. This extensive recombinant protein repository provides a powerful tool for proteome- and genome-scale research of this organism.

PMID:
20359537
DOI:
10.1016/j.pep.2010.03.019
[Indexed for MEDLINE]
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