Secretion of heterologous protein in yeast using glucoamylase signal sequence of saccharomyces diastaticus

Abstract

For the purpose of investigating the potential of the glucoamylase signal sequence to construct a yeast secretion system to secrete useful heterologous proteins, the secretions of Bacillus stearothermophilus α-amylase and human lipocortin-1 have been attempted using glucoamylase signal sequence in yeast Saccharomyces sp. As a preliminary study to optimize the glucoamylase signal sequence for the construction of a secretion vector system, two cloned genes, STA1 and SGA coding for the extracellular glucoamylase and sporulation-specific glucoamylase, respectively, were analyzed by Southern blot and nucleotide sequencing analysis and the role of Thr- and Ser-rich region (TS region) of STA1 in the secretion of endo-1,4-β-D-glucanase (carboxymethyl cellulase or CMCase) from Bacillus subtilis was investigated. According to the results of Southern blot analysis, the promoter and signal sequence of STA1 was very homologous to 1.5-kb BamHI DNA fragment (S2), and TS region and mature enzyme-coding region of STA1 had a high homology to 7.5-kb BamHI fragment (S1) and SGA, respectively. The determination of nucleotide sequences revealed that amino acid sequcnces deduced from nucleotide sequences of SGA were almost the same as those of the mature enzyme-coding region of STA1. The probable cleavage site for signal peptidase was also predicted by calculating S-value for each amino acid residue of N-terminal region, suggesting $Gly^{21}$ was a cleavage site. For the secretion of B. stearothermophilus α-amylase from yeast, a recombinant plasmid pGAT17 was constructed by fusing B. stearothermophilus α-amylase structural gene in frame to the promoter and signal sequence of Saccharomyces diastaticus glucoamylase gene (STA1). The secretion of the heterologous α-amylase from S. diastaticus transformed with pGAT17 was confirmed by the halo formation around colonies on selective starch agar medium. About 80% of the total α-amylase activity was detected in the growth medium. The secre...