caa a22 4500 475744411 CHVBK 20180406123509.0 cr unu---uuuuu 170329e20001101xx s 000 0 eng 10.1023/A:1007148202270 doi (NATIONALLICENCE)springer-10.1023/A:1007148202270 A Functional Raw Starch-Binding Domain of Barley α-Amylase Expressed in Escherichia coli [Elektronische Daten] [Brian Tibbot, Dominic Wong, George Robertson] The mature form of barley seed low-pI α-amylase (BAA1) possesses a raw starch-binding site in addition to the catalytic site. A truncated cDNA encoding the C-terminal region (aa 281-414) and containing the proposed raw starch-binding domain (SBD) but lacking Trp278/Trp279, a previously proposed starch granule-binding site, was synthesized via PCR and expressed in Escherichia coli as an N-terminal His-Tag fusion protein. SBD was produced in the form of insoluble inclusion bodies that were extracted with urea and successfully refolded into a soluble form via dialysis. To determine binding, SBD was purified by affinity chromatography with cycloheptaamylose as ligand cross-linked to Sepharose. This work demonstrates that a SBD is located in the C-terminal region and retains sufficient function in the absence of the N-terminal, catalytic, and Trp278/279 regions. Plenum Publishing Corporation, 2000 Amylase nationallicence barley nationallicence carbohydrate binding nationallicence cyclodextrin nationallicence raw starch nationallicence recombinant protein nationallicence Tibbot Brian Western Regional Research Center, USDA-ARS, 800 Buchanan Street, 94710, Albany, California aut Wong Dominic Western Regional Research Center, USDA-ARS, 800 Buchanan Street, 94710, Albany, California aut Robertson George Western Regional Research Center, USDA-ARS, 800 Buchanan Street, 94710, Albany, California aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/8(2000-11-01), 663-669 0277-8033 19:8<663 2000 19 10930 https://doi.org/10.1023/A:1007148202270 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1007148202270 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Tibbot Brian Western Regional Research Center, USDA-ARS, 800 Buchanan Street, 94710, Albany, California aut NATIONALLICENCE 700 1- Wong Dominic Western Regional Research Center, USDA-ARS, 800 Buchanan Street, 94710, Albany, California aut NATIONALLICENCE 700 1- Robertson George Western Regional Research Center, USDA-ARS, 800 Buchanan Street, 94710, Albany, California aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/8(2000-11-01), 663-669 0277-8033 19:8<663 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer