To increase the production of keratinase, stable strains of Bacillus licheniformis carrying multiple keratinase gene copies in the chromosome were developed. Integrative vectors carrying kerA with or without P43-promoter were constructed and subcloned into B. licheniformis T399D and Bacillus subtilis DB104. In T399D, multiple copies of kerA integration into the chromosome were identified and determined by Southern blot. The optimal integration of kerA was found in the range of 3-5 copies. Higher integration of gene copies (>5) caused reduced processing and secretion of the extracellular keratinase. In DB104, kerA was cloned in the plasmid, not integrated into the chromosome. The strong constitutive promoter P43 not only increased the keratinase production in plasmid-based expression in DB104 but also improved the enzyme yield of the integrants of T399D. New strains were able to enhance cell growth and enzyme yield at higher concentrations of medium substrate. When they were grown in either soy or feather medium, the keratinase activity was stable and improved by about 4-6 times.
Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrP Sc , is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrP Sc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease-causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion-like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.Keywords: BSE; prion; PrP Sc ; Sup35NM; yeast prion; prion surrogate protein; enzymatic degradation Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a unique group of fatal neurodegenerative disorders found in humans and animals (Prusiner et al. 1998). They include diseases such as kuru and Creutzfeld-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE, widely known as mad cow disease) in cattle (Parchi and Gambetti 1995), and TSEs in other mammals (Burger and Hartsough 1965;Williams and Young 1980;Leggett et al. 1990). All of these diseases are characterized by extensive neuronal loss giving brain tissue a spongy appearance.The pathological isoform of prion protein (PrP Sc ) aggregates in diseased tissues and, in common conditions, is resistant to digestion by proteases (Caughey et al. 1991;Pan et al. 1993). It was recently found that PrP Sc in diseased tissues can be degraded by a feather-degrading keratinase and other microbial proteases when the tissue homogenate was preheated at 115 C (Langeveld et al. 2003). Independently, mouse bioassays showed a significant reduction of infectivity after a proteolytic inactivation process (McLeod et al. 2004). These results indicated a potential method of enzymatic inactivation of prion protein.PrP Sc is pathogenic and dangerous to handle. A mutant recombinant prion produced in Escherichia coli without glycosylation was recently found to be able to induce neurologic dysfunction (Legname et al. 2004). Experimentation with prion tissues must be strictly controlled and is inordinately expensive. A nonpathogenic prion surrogate protein which has similar physical-chemical properties Reprint requests to: Ching-Ying Chen, Department of Poultry Science, North Carolina State University, Raleigh, NC 27695-7608, USA; e-mail: cchen8@ncsu.edu; fax: (919) 515-2625.Article ...
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