Enhanced secretion of<Emphasis Type="Italic"> Bacillus stearothermophilus</Emphasis> L1 lipase in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> by translational fusion to cellulose-binding domain |
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Authors: | J?O?Ahn E?S?Choi H?W?Lee S?H?Hwang C?S?Kim H?W?Jang S?J?Haam Email author" target="_blank">J?K?JungEmail author |
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Institution: | (1) Bio-Pilot Plant, Korea Research Institute of Bioscience and Biotechnology, Yusong, 305-600 Taejon, Korea;(2) Laboratory of Microbial Functions, Korea Research Institute of Bioscience and Biotechnology, Yusong, 305-600 Taejon, Korea;(3) Department of Chemical Engineering, Yonsei University, Sodaemun-ku, 120-749 Seoul, Korea;(4) Acebiotech Corporation, Chungwon, 363-942 Chungbuk Province, Korea |
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Abstract: | The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the fusion protein in S. cerevisiae was constructed using the -amylase signal peptide and the galactose-inducible GAL10 promoter. Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion. The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures. Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus. Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S. cerevisiae, since in vivo breakage at the linker was almost negligible. |
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