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.