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

Protein Expr Purif. 2003 Aug;30(2):185-93. doi: 10.1016/s1046-5928(03)00103-7.


Barley alpha-amylase/subtilisin inhibitor (BASI) is a 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 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-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 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics*
  • Hordeum
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / metabolism
  • Plant Proteins / antagonists & inhibitors
  • Plant Proteins / biosynthesis*
  • Plant Proteins / isolation & purification*
  • Plant Proteins / metabolism
  • Plant Proteins / pharmacology
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Surface Plasmon Resonance
  • Trypsin Inhibitor, Kunitz Soybean / biosynthesis*
  • Trypsin Inhibitor, Kunitz Soybean / genetics
  • Trypsin Inhibitor, Kunitz Soybean / isolation & purification*
  • Trypsin Inhibitor, Kunitz Soybean / pharmacology
  • alpha-Amylases / antagonists & inhibitors
  • alpha-Amylases / metabolism


  • Isoenzymes
  • Plant Proteins
  • Recombinant Proteins
  • barley alpha-amylase-subtilisin inhibitor
  • Trypsin Inhibitor, Kunitz Soybean
  • AMY1.2 protein, Hordeum vulgare
  • alpha-Amylases