Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli

• Published in: Protein expression and purification Vol. 44; no. 2; pp. 130 - 135
• Main Authors: Shimizu, Tetsuya, Shibata, Hiroyuki, Araya, Tomoyuki, Nakatsu, Toru, Miyairi, Kazuo, Okuno, Toshikatsu, Kato, Hiroaki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2005
• Subjects: Agaricales - enzymology; Amino Acid Sequence; Blotting, Western; Crystallization; Crystallography, X-Ray; Disulfide bond; Electrophoresis, Polyacrylamide Gel; Endopolygalacturonase; Escherichia coli; Escherichia coli - genetics; Molecular Weight; Oxidizing bacterial cytoplasm; Pectins - chemistry; Plasmids - genetics; Polygalacturonase - genetics; Polygalacturonase - isolation & purification; Polygalacturonase - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Spectrometry, Mass, Electrospray Ionization; Stereum purpureum; Endopolygalacturonase; Disulfide bond; Oxidizing bacterial cytoplasm; Crystallization
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2005.06.001

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the α-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 Å resolution with synchrotron radiation.