Expression and purification of a trefoil peptide motif in a beta-galactosidase fusion protein and its use to search for trefoil-binding sites

Eur J Biochem. 1993 Mar 1;212(2):557-63. doi: 10.1111/j.1432-1033.1993.tb17693.x.


The cysteine-rich trefoil motif of rat intestinal trefoil factor (rITF) was cloned and expressed in Escherichia coli. A 270-bp cDNA fragment including the signal sequence and the trefoil motif was cloned into the expression vector pAX5+ to direct the expression of a beta-galactosidase collagen-hinged fusion protein in E. coli. Cultures harbouring the recombinant plasmid produced a soluble novel protein with a molecular mass of 134.5 kDa, as predicted for the trefoil-motif-containing fusion protein. Purification of the rITF moiety was achieved by p-aminophenyl-thio-beta-D-galactoside(APTG)-affinity chromatography, collagenase digestion of the hybrid molecule, and removal of the beta-galactosidase-hinge molecule by a further APTG-affinity step. It was demonstrated that intrachain disulphide-bond formation in rITF occurred during the procedure, so no refolding steps were required. Analysis by immunoblotting revealed that the fusion protein and the cleaved trefoil-motif-containing protein were recognised by an antibody raised against the chemically synthesised peptide. The trefoil motif present in the fusion protein was used to localise putative trefoil-binding sites in sections of frozen rat tissue. Binding was demonstrated using the beta-galactosidase portion of the fusion protein as a reporter moiety, either directly with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside, or indirectly using a monoclonal antibody to beta-galactosidase and indirect immunohistochemistry. Binding sites were localised to the foveolar and surface epithelium of rat stomach, the collecting ducts of the kidney and within colonic crypts. The presence of a trefoil motif was necessary for binding. The use of beta-galactosidase fusion proteins for histochemical localisation of peptide-binding sites should prove more generally useful.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • Cloning, Molecular
  • Escherichia coli / metabolism
  • Growth Substances / biosynthesis
  • Growth Substances / genetics
  • Growth Substances / metabolism*
  • Immunoblotting
  • Molecular Sequence Data
  • Mucins*
  • Muscle Proteins*
  • Neuropeptides*
  • Peptides*
  • RNA, Messenger / analysis
  • Rats
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism*
  • Trefoil Factor-2
  • Trefoil Factor-3
  • beta-Galactosidase / metabolism*


  • Growth Substances
  • Mucins
  • Muscle Proteins
  • Neuropeptides
  • Peptides
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • TFF3 protein, rat
  • Trefoil Factor-2
  • Trefoil Factor-3
  • beta-Galactosidase

Associated data