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 en coding the C-terminal region (aa 281-414) and containing the proposed raw s tarch-binding domain (SBD) but lacking Trp278/Trp279, a previously proposed starch granule-binding site, was synthesized via PCR and expressed in Esch erichia coli as an N-terminal His-Tag fusion protein. SBD was produced in t he form of insoluble inclusion bodies that were extracted with urea and suc cessfully refolded into a soluble form via dialysis. To determine binding, SBD was purified by affinity chromatography with cycloheptaamylose as ligan d cross-linked to Sepharose. This work demonstrates that a SBD is located i n the C-terminal region and retains sufficient function in the absence of t he N-terminal, catalytic, and Trp278/279 regions.