A functional raw starch-binding domain of barley alpha-amylase expressed in Escherichia coli.
The mature form of barley seed low-pI alpha-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.
Tibbot
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BK
Western Regional Research Center, USDA-ARS, Albany, California 94710, USA.
Wong
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DW
Robertson
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GH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D001467:Hordeum / Q000201:enzymology*; D008969:Molecular Sequence Data; D013213:Starch / Q000378:metabolism*; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D013213:Starch; D000516:alpha-Amylases