Enzymic preparation of protein G-peroxidase conjugates catalysed by transglutaminase.
Transglutaminases (TGases, EC 2.3.2.13) have proved to be valuable enzymes for site-directed protein coupling via N(epsilon)-(gamma-L-glutamyl)lysine bonds. Their use in conjugate synthesis would overcome many problems caused by chemical reagents. In this approach, we show for the first time that two proteins with different functionalities, namely soybean peroxidase and protein G, can be cross-linked by bacterial TGase with retention of their activities. Soybean peroxidase and protein G were chosen for the enzymic preparation of a bifunctional conjugate among a series of other TGase substrates detected by enzymic incorporation of small fluorescent or biotinylated molecules. The highest yields of conjugate were obtained with a 15-fold excess of peroxidase in phosphate buffer, pH 7.0. Size exclusion chromatography enabled both purification of the conjugates and recovery of the starting materials. Analysis of bifunctionality revealed the coupling of protein G with an average of three peroxidase molecules.
Bechtold
U
U
Fachbereich Chemische Technologie, Fachhochschule Darmstadt, Darmstadt, Germany.
Otterbach
J T
JT
Pasternack
R
R
Fuchsbauer
H L
HL
D002103:Cadaverine / Q000031:analogs & derivatives / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D005973:Glutamine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013449:Sulfonamides / Q000737:chemistry; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
C108034:1-N-(carbobenzoxy-glutaminylglycyl)-5-N-(5'-N',N'-dimethylamino-1'-naphthalenesulfonyl)diamidopentane; D004151:Dipeptides; D005456:Fluorescent Dyes; C029152:G-substrate; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D013449:Sulfonamides; D005973:Glutamine; D009195:Peroxidase; D011503:Transglutaminases; C008542:monodansylcadaverine; D008239:Lysine; D002103:Cadaverine