纯化纤维素【荐】.pdfVIP

Biotechnol. Appl. Biochem. (2000) 31, 197–203 (Printed in Great Britain) 197 Expression, purification and applications of staphylococcal Protein A fused to cellulose-binding domain Etai Shpigel*, Arie Goldlust, Adi Eshel, Idit Kaplan Ber*, Gilat Efroni, Yossi Singer, Ilan Levy*, Mara Dekel* and Oded Shoseyov*1 *The Kennedy Leigh Centre for Horticulture Research and The Otto Warburg Center for Agricultural Biotechnology, The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel, and CBD-Technologies Ltd., Park Tamar, P.O. Box 199, Rehovot 76100, Israel Because staphylococcal Protein A (ProtA) binds Efficient immobilization of ProtA is required for most of specifically to IgG, it has been used for many immuno- these applications, and various methods of immobilization logical manipulations, most notably antibody puri- have been developed in recent years. Most of them require fication and diagnostics. Immobilization is required for chemical modifications of the matrix. These modifications, most of these applications. Here we describe a genetic- leading to covalent bonding of the ligand to the matrix, result engineering approach to immobilizing ProtA on cellu- in many cases in loss of activity of the ligand as well as the lose, by fusing it to cellulose-binding domain (CBD) inclusion of toxic organic compounds that must be removed derived from the cellulose-binding Protein A of Clos- before use. A genetic engineering approach has been used to tridium cellulovorans. The bifunctional fusion protein overcome some of these problems by fusing different affinity was expressed in Escherichia coli, recovered o