Cloning and heterologous expression of gene encoding A polygalacturonase from Aspergillus awamori.
A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.
Nagai
M
M
Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University, Nakamachi, Nara, Japan.
Ozawa
A
A
Katsuragi
T
T
Kawasaki
H
H
Sakai
T
T
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007438:Introns; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011096:Polygalacturonase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae
D011096:Polygalacturonase