title: Expression of cDNAs encoding barley alpha-amylase 1 and 2 in yeast and characterization of the secreted proteins.
abstract: Amylolytic strains of the yeast, Saccharomyces cerevisiae, were constructed by transformation with expression plasmids containing cDNAs encoding either AMY1 (clone E) or AMY2 (clone pM/C). The alpha-amylases were efficiently secreted into the culture medium directed by their own signal peptides. When clone E without its 5'-noncoding region was expressed from the yeast PGK promoter, AMY1 was produced as 1% of total cell protein and was thus the major protein secreted, whereas a similar construct derived from pM/C produced much less AMY2. This level is the highest reported for a plant protein secreted by yeast as mediated by the endogenous signal peptide. Production of AMY1 increased 25-fold when the 5'-noncoding part of clone E which contains a 12-bp dG.dC homopolymer tail had been removed. Moreover, expression was one to two orders of magnitude higher when genes encoding AMY1 or AMY2 were inserted between promoter and terminator of the yeast PGK gene in comparison to expression directed from the ADC1 or GAL1 promoters. Recombinant AMY1 and AMY2 had the same Mr and N-terminal sequence as the corresponding barley malt enzymes. Furthermore, none of the enzymes were found to be N-glycosylated. Isoelectric focusing indicated that transformed yeast cells secreted one major form of AMY2 and four dominant forms of AMY1. One AMY1 form corresponded to one of the major forms found in malt while the others, having either low activity or unusually high pI, probably reflect inefficient/incorrect processing. Enzyme kinetic properties and pH activity-dependence of recombinant AMY2 were essentially identical to those of malt AMY2.(ABSTRACT TRUNCATED AT 250 WORDS)
authors: Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark.
authors: M
authors: M
authors: Søgaard
authors: B
authors: B
authors: Svensson
mesh_terms: D015153:Blotting, Western; D004247:DNA / Q000096:biosynthesis; D015966:Gene Expression Regulation, Fungal; D001467:Hordeum / Q000235:genetics*; D007525:Isoelectric Focusing; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D004247:DNA; D000516:alpha-Amylases