Expression and purification of a trefoil peptide motif in a beta-galactosidase fusion protein and its use to search for trefoil-binding sites.
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.
Chinery
R
R
Imperial Cancer Research Fund/Royal College of Surgeons Histopathology Unit, London, England.
Poulsom
R
R
Elia
G
G
Hanby
A M
AM
Wright
N A
NA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006133:Growth Substances / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009077:Mucins; D009124:Muscle Proteins; D009479:Neuropeptides; D010455:Peptides; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3; D001616:beta-Galactosidase / Q000378:metabolism*
D006133:Growth Substances; D009077:Mucins; D009124:Muscle Proteins; D009479:Neuropeptides; D010455:Peptides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C099037:TFF3 protein, rat; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3; D001616:beta-Galactosidase