Cloning and heterologous expression of gene encoding a polygalacturonase from Aspergillus awamori

Citation
M. Nagai et al., Cloning and heterologous expression of gene encoding a polygalacturonase from Aspergillus awamori, BIOS BIOT B, 64(8), 2000, pp. 1580-1587
Citations number
25
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Agricultural Chemistry","Biochemistry & Biophysics
Journal title
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
ISSN journal
0916-8451 → ACNP
Volume
64
Issue
8
Year of publication
2000
Pages
1580 - 1587
Database
ISI
SICI code
0916-8451(200008)64:8<1580:CAHEOG>2.0.ZU;2-F
Abstract
A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by geno mic Southern hybridization with a probe of a DNA fragment synthesized by PC R. This was done using primers constructed based on the N-terminal amino ac id sequence of a polygalacturonase, protopectinase-AS, produced by the stra in and the consensus internal amino acid sequence of fungal polygalacturona ses. The cloned polygalacturonase gene, containing an ORF, encodes 362 amin o acids, including a 52-bp intron. It contains the consensus nucleotide seq uence of PacC binding sites, and its expression was appeared to be regulate d by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharom yces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase p roduced was the product of the cloned gene.