Subtilisins and other serine proteases are extensively used in the detergent, leather and food industry, and frequently under non-physiological conditions. New proteases with improved performance at extreme temperatures and in the presence of chemical additives may have great economical potential. The increasing availability of genetic sequences from different environments makes homology-based screening an attractive strategy for discovery of new proteases. A prerequisite for large-scale screening of protease-encoding sequences is an efficient screening procedure. We have developed and implemented a screening procedure that encompasses cloning of candidate sequences into multiple expression vectors, cytoplasmic... More
Subtilisins and other serine proteases are extensively used in the detergent, leather and food industry, and frequently under non-physiological conditions. New proteases with improved performance at extreme temperatures and in the presence of chemical additives may have great economical potential. The increasing availability of genetic sequences from different environments makes homology-based screening an attractive strategy for discovery of new proteases. A prerequisite for large-scale screening of protease-encoding sequences is an efficient screening procedure. We have developed and implemented a screening procedure that encompasses cloning of candidate sequences into multiple expression vectors, cytoplasmic expression in E. coli, and a casein-based functional screen. The procedure is plate-format compatible and can be completed in only four days, starting from the gene of interest in a suitable cloning vector. The expression vector suite includes six vectors with combinations of maltose-binding protein (MBP) or the small ubiquitin-related modifier (SUMO) for increased solubility, and polyhistidine tags for downstream purification. We used enhanced green fluorescent protein and four Bacilli subtilisins to validate the screening procedure and our results show that proteins were expressed, soluble and active. Interestingly, the highest activities were consistently achieved with either MBP or SUMO fusions, thus demonstrating the merit of including solubility tags. In conclusion, the results demonstrate that our approach can be used to efficiently screen for new subtilisins, and suggest that the approach may also be used to screen for proteins with other activities.
