Monday, November 5, 2007
97-2
Release and Binding of Recombinant Proteins Produced by Transgenic Tobacco on Montmorillonite and Kaolinite, and Microbial Utilization and Enzymatic Activity of Free and Clay-Bound Proteins.
Human serum albumin (HSA), ß-glucuronidase (GUS), glycoprotein B (gB) from cytomegalovirus, and green fluorescent protein (GFP) are expressed by genetically-modified tobacco. The release of these proteins in root exudates of transgenic tobacco in sterile hydroponic culture and nonsterile soil was determined. GUS, gB, and GFP were not released in root exudates, whereas HSA was, as shown by a 66.5-kDa band on SDS-PAGE and Western blots and confirmed by enzyme-linked immunosorbent assay (ELISA). HSA and GUS bound rapidly on the clay minerals, kaolinite (K) and montmorillonite (M). Adsorption increased as the concentration of protein increased and then reached a plateau. More adsorption and binding occurred with GUS: 2.2±0.29 µg adsorbed and 1.7±0.21 µg bound µg-1 of M; 1.5±0.28 µg adsorbed and 1.0±0.03 µg bound µg-1 of K. With HSA: 1.2±0.04 µg adsorbed and 0.8±0.05 µg bound µg-1 of M; 0.4±0.05 µg adsorbed and 0.4±0.03 µg bound µg-1 of K. However, only HSA intercalated M, as shown by X-ray diffraction analyses. None of the proteins intercalated K, a nonswelling clay. When bound, the proteins were not utilized for growth by mixed cultures of soil microorganisms, whereas the cultures readily utilized the free (i.e., not bound) proteins as sources of carbon and energy. The enzymatic activity of GUS was significantly enhanced when bound on the clay minerals. These results indicated that recombinant proteins expressed by transgenic plants could persist and function in soil after their release in root exudates and from decaying plant residues as the result of the protection provided against biodegradation by binding on clay minerals.
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