Mycobacterial proteome extraction: Comparison of disruption methods

Abstract

Mycobacterium avium subsp. paratuberculosis has long been recognized as the causative agent of Johne's disease, a chronic inflammatory intestinal disease of sheep, cattle and other ruminants. Mycobacterial cells are extremely hardy, and proteomic analyses require the use of harsh conditions to effect their disruption. We compared the effectiveness of bead beating and sonication as cell lysis methods for the extraction of the proteomes of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis. Broad and narrow range two-dimensional gel electrophoresis was used to compare the numbers of silver stained protein spots that were observed in mycobacterial lysates. Despite differences in the yield of total protein from either species, and at different ages, the two methods appeared to give similar representations of the mycobacterial proteomes analyzed. Bead beating therefore represents a rapid and effective method of extracting the proteomes of mycobacterial species without the risks associated with an open tube sonication procedure.

Proteomic comparison of Mycobacterium avium subspecies paratuberculosis grown in vitro and isolated from clinical cases of ovine paratuberculosis

“…This had a significant impact on the quality of the proteomic profile generated, making comparisons easier. Electrophoretograms generated by our procedure compared well with those generated by the recently published method of Lanigan et al (2004) for 2D gel electrophoresis of M. avium subsp. paratuberculosis and M. avium subsp.…”

“…This was consistent with the presence of contaminating nonproteinaceous impurities, and can be seen elsewhere in M. avium subsp. paratuberculosis 2D protein profiles (Lanigan et al, 2004). By employing the extraction and clean-up procedures described above, we succeeded in reducing the horizontal streaking to a minimum, enabling subsequent gel comparison.…”

Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction

“…However, 10,14,20 Another cause for reduced sensitivity and increased false-negative results is the inadequate cell lysis of MAP, due to the characteristics of the MAP cell wall. 11 The objective of the current study was to evaluate the ability of 6 different commercial fecal DNA extraction kits to extract DNA from MAP present in feces.…”

Elucidating the Mechanism of Vanillin Induced Mycobacterial Membrane Disruption: Implications of Lipid Alteration

“…smegmatis cells were grown in Middlebrook 7H9 broth in the absence (control) and presence of Van (15μg/ml). Whole cell protein was extracted [13] and protein concentration was determined by Lowry method as previously described [14]. Lipase activity was performed by measuring the amount of p-nitrophenol (p-NP) released from p-NP ester substrate with varying lengths of fatty acids.…”