Monitoring the transport of recombinant Candida rugosa lipase by a green fluorescent protein-lipase fusion.
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.
Passolunghi
Simone
S
Dipartimento di Biotecnologie e Bioscienze, Universita' degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Brocca
Stefania
S
Cannizzaro
Luca
L
Porro
Danilo
D
Lotti
Marina
M
D002175:Candida / Q000201:enzymology* / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D049452:Green Fluorescent Proteins; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008049:Lipase