title: Enhanced secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae by translational fusion to cellulose-binding domain.
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 alpha-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.
authors.affiliation: Bio-Pilot Plant, Korea Research Institute of Bioscience and Biotechnology, Yusong, 305-600 Taejon, Korea.
authors.forename: J O
authors.initials: JO
authors.lastname: Ahn
authors.forename: E S
authors.initials: ES
authors.lastname: Choi
authors.forename: H W
authors.initials: HW
authors.lastname: Lee
authors.forename: S H
authors.initials: SH
authors.lastname: Hwang
authors.forename: C S
authors.initials: CS
authors.lastname: Kim
authors.forename: H W
authors.initials: HW
authors.lastname: Jang
authors.forename: S J
authors.initials: SJ
authors.lastname: Haam
authors.forename: J K
authors.initials: JK
authors.lastname: Jung
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D043484:Proprotein Convertases / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D013997:Time Factors; D014242:Trichoderma
chemical_list: D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D002482:Cellulose; D008049:Lipase; C056373:endoglucanase 2; D002480:Cellulase; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae