Monitoring the transport of recombinant Candida rugosa lipase by a green fluorescent protein-lipase fusion

Author： Passolunghi Simone   Brocca Stefania   Cannizzaro Luca   Porro Danilo   Lotti Marina  

Publisher： Springer Publishing Company

ISSN： 0141-5492

Source： Biotechnology Letters, Vol.25, Iss.22, 2003-11, pp. : 1945-1948

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Abstract

In a batch cultivation of Pichia pastoris expressing Candida rugosa lipase 1 (CRL1), secretion of 200 μg 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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