Monitoring the transport of recombinant Candida rugosa lipase by a green fluorescent protein-lipase fusion |
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Authors: | Simone Passolunghi Stefania Brocca Luca Cannizzaro Danilo Porro Marina Lotti |
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Institution: | Dipartimento di Biotecnologie e Bioscienze, Universita' degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy. |
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Abstract: | In a batch cultivation of Pichia pastoris expressing Candida rugosa lipase 1 (CRL1), secretion of 200 microg lipase ml(-1) of culture was achieved in sorbitol-based medium. However, a large amount of recombinant protein was retained intracellularly throughout the fermentation, pointing to the transport step as a major bottleneck. Therefore a translational fusion with the green fluorescent protein (GFP) was constructed that was expressed and transported similarly to the native lipase and retained catalytic activity. This analytical tool enables a rapid monitoring of product localization and amount, based on GFP-associated fluorescence. |
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Keywords: | fluorimetry green fluorescent protein lipase Pichia pastoris secretion |
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