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.

Thermococcus kodakarensis as a Host for Gene Expression and Protein Secretion

“…With yeast host cells, 25 to 30 mg protein g DCW Ϫ1 have been reported for Saccharomyces cerevisiae (1,7,25), while 2 to 4 mg protein g DCW Ϫ1 have been reported for Pichia pastoris (6,20). It is often the case that large differences in production levels are observed using the same host cell, presumably due to differences in promoters, culture conditions, and the protein being expressed.…”

Strategy for selecting and characterizing linker peptides for CBM9‐tagged fusion proteins expressed in Escherichia coli

“…The technology is based on genetically linking a short peptide or small protein domain to either the aminoor carboxy-terminus of the target protein and then using the fusion tag to facilitate efficient capture and purification of the chimeric protein on an affinity matrix. Certain fusion partners such as the maltose binding protein (MBP) (di Guan et al, 1988), carbohydrate binding modules (CBMs) (Ahn et al, 2004;Murashima et al, 2003), thioredoxin (Trx) (LaVallie et al, 1993), and the Escherichia coli protein N-utilizing substance A (NusA) (Davis et al, 1999), have been shown to improve product solubility and expression by stabilizing the mRNA (Makrides, 1996) or by providing a psuedo-chaperone effect (Samuelsson et al, 1994). Fusion tags have also been used to increase in vivo proteolytic stability (Jansson et al, 1990;Martinez et al, 1995;Staahl and Nygren, 1997), to serve as effective expression and localization reporters (Gerdes and Kaether, 1996;Ozawa, 2006), and, when combined with their corresponding leader peptide, to control product localization in or secretion from the expression host (Ford et al, 1991;Johnson et al, 1992;Uhlen and Moks, 1990).…”

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

“…Recent studies have shown that a CBM serving as a fusion partner may have additional values. In the expression of CBM-lipase fusion protein in yeast, for example, it was shown that CBM also enhanced secretion (1).…”