DIRECT EXTRACTION OF PROTEINS FROM ENVIRONMENTAL-SAMPLES

Two methods were developed and evaluated for extracting proteins from water, sediment, and soil samples. In the first method, microbial proteins were extracted from 1 g soil or sediment, or pellet from 10 ml wastewater, by boiling the samples in a solution containing 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 10% glycerol, and 0.2% bromophenol blue. In the second method, the same quantities of environmental samples were incubated for 1 h at 0-degrees-C in a 2 volume solution containing 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 10% sucrose, 1 mM dithiothreitol, 300 mug/ml lysozyme, 0.1% polyoxyethylene 20 cetyl ether. Lysis was completed by four 10-min freeze-thaw cycles (37-degrees-C to dry-ice bath). Cellular debris and other particulate matter were removed from the protein preparations by centrifugation. The boiling method recovered high concentrations of proteins from wastewater (10 to 30 mug per ml), but not from soils and sediments. The freeze-thaw method performed better for soils and sediments, yielding 20 to 50 mug protein per g. Protein extracts were resolved by electrophoresis using 15% polyacrylamide gels under denaturing conditions. The sizes of proteins extracted by both methods ranged from less than 14 kDa to greater than 97 kDa. Both methods are simple and rapid, and both have potential applications for direct analysis of microbial community response to changes in environmental conditions, and for direct measurement of gene expression in complex microbial communities.