Purification and characterization of the β-trefoil fold protein barley α-amylase/subtilisin inhibitor overexpressed in Escherichia coli

Barley alpha-amylase/subtilisin inhibitor (BASI) is beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mg L His(6)-rBASI; (ii) 6mgL(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa-, (iii) 3 mg L-1 untagged rBASI; and (iv) 0.2 mg L-1 rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mg L-1. The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K-i of 0.10, 0.06, and 0.09nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on) = 1.3 x 10(5) M-1 s(-1), k(off) = 1.4 x 10(-4) s(-1), and K-D = 1.1 nM, and of the savinase-His(6)-rBASI complex k(on) = 21.0 x 10(4) M-1 s(-1), k(off) = 53.0 x 10(-4) s(-1), and K-D = 25.0nM, in agreement with sBASI values. Ki was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively. (C) 2003 Elsevier Science (USA). All rights reserved.