Quintero David, Bermudes David
Department of Biology, California State University Northridge, Northridge, CA 91330-8303, USA.
Department of Biology, California State University Northridge, Northridge, CA 91330-8303, USA; Interdisciplinary Research Institute for the Sciences (IRIS), California State University Northridge, Northridge, CA 91330-8303, USA.
J Microbiol Methods. 2014 May;100:105-10. doi: 10.1016/j.mimet.2014.02.019. Epub 2014 Mar 12.
We have developed a culture-based method for determining the production of secreted protease inhibitors. The assay utilizes standard proteolysis detection plates to support microbial growth followed by infiltrating the plate with a protease and subsequently detecting the remaining protein by trichloroacetic acid (TCA) precipitation, or by bromocreosol green (BCG) or Ponseau S (PS) staining. The presence of a protease inhibitor can be observed in the form of a protected zone of protein around the protease inhibitor-producing strain. Using the protease inhibitors α-2-macroglobulin, aprotinin, leupeptin, and bestatin and the primary and secondary forms of Photorhabdus luminescens in combination with the protease trypsin, we were able to demonstrate that the assay is specific for the cognate inhibitor of the protease and for bacteria secreting protease inhibitors. In addition, when casein-containing plates were used, the size of the diffusion zone was inversely correlated with the molecular weight of the inhibitor allowing a relative estimation of the protease inhibitor molecular weight. This assay is useful for detecting the presence of microbial secreted protease inhibitors and may reveal their production by microorganisms that were not previously recognized to produce them.
我们开发了一种基于培养的方法来测定分泌型蛋白酶抑制剂的产生。该测定法利用标准的蛋白水解检测板来支持微生物生长，随后用蛋白酶渗透平板，然后通过三氯乙酸（TCA）沉淀、溴甲酚绿（BCG）或丽春红S（PS）染色来检测剩余的蛋白质。蛋白酶抑制剂的存在可以以产生蛋白酶抑制剂的菌株周围的蛋白质保护区域的形式观察到。使用蛋白酶抑制剂α-2-巨球蛋白、抑肽酶、亮抑酶肽和贝司他汀以及发光杆菌的初级和次级形式与胰蛋白酶结合，我们能够证明该测定法对蛋白酶的同源抑制剂和分泌蛋白酶抑制剂的细菌具有特异性。此外，当使用含酪蛋白的平板时，扩散区的大小与抑制剂的分子量呈负相关，从而可以相对估计蛋白酶抑制剂的分子量。该测定法可用于检测微生物分泌的蛋白酶抑制剂的存在，并可能揭示以前未被认为能产生它们的微生物产生这些抑制剂的情况。
