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United States Department of Agriculture

Agricultural Research Service

Research Project: TESTING OF RECOMBINANT ANTIGENS FOR THE SERODIAGNOSIS OF JOHNE'S DISEASE Title: Characteristics of an Extensive Mycobacterium avium subspecies paratuberculosis Recombinant Protein Set

Authors
item Bannantine, John
item Stabel, Judith
item Bayles, Darrell
item Giesbrecht, Brian -

Research conducted cooperatively with:
item

Submitted to: Protein Expression and Purification
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 17, 2010
Publication Date: August 1, 2010
Repository URL: http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WPJ-4YR8RFR-1-3&_cdi=6992&_user=4250360&_pii=S104659281000094X&_orig=search&_coverDate=03%2F30%2F2010&_sk=999999999&view=c&wchp=dGLzVlb-zSkzV&md5=509a70f52df41b22e2ea8537aad68f59&ie=/sdarticle.pdf
Citation: Bannantine, J.P., Stabel, J.R., Bayles, D.O., Geisbrecht, B. 2010. Characteristics of an Extensive Mycobacterium avium subspecies paratuberculosis Recombinant Protein Set. Protein Expression and Purification. 72(2):223-233.

Interpretive Summary: This manuscript reports on the catalog of over 600 recombinant proteins produced for Mycobacterium avium subspecies paratuberculosis. It discusses unique characteristics of certain categories of proteins and looks at the collection as a whole. The information from this manuscript is of interest to scientists working in this and related fields. This manuscript demonstrates completion of milestone 1.2 through FY2010 in the current Johne's Disease Research(3625-32000-093-00D).

Technical Abstract: In the first step of a comprehensive large-scale antigen discovery project, 651 Mycobacterium avium subspecies paratuberculosis proteins were produced 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 PSORTb predicted membrane proteins, totaling 252, resulted in lower average expression yields (3.51 mg/L culture) than the 176 predicted cytoplasmic proteins (7.27 mg/L culture). Some genes could not be cloned and several others would not express protein. A few proteins (MAP0107c, MAP3169c and MAP3640) appear to promote lysis of E. coli since there was an immediate drop in optical density 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, a defined set of 19 proteins that showed an absorbance maximum at 260nm rather than 280nm were identified. This extensive recombinant protein repository provides a powerful tool for proteome- and genome-scale research of this organism.

Last Modified: 7/31/2014