A versatile method for the coupling of protein to DNA: synthesis of alpha 2-macroglobulin-DNA conjugates.
We describe a simple, general method to link proteins covalently to DNA. The method uses two reagents, N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine and 2-iminothiolane. The former reacts specifically with nonpaired quanine residues and upon reduction generates a free sulfhydryl group. The latter reacts with a protein to provide another sulfhydryl group which is subsequently conjugated to DNA by an intermolecular disulfide interchange reaction. Using this method alpha 2-macroglobulin was conjugated to plasmid DNA encoding the Herpes simplex virus-1 thymidine kinase gene or a DNA fragment containing the E. coli chloramphenicol acetyltransferase gene. Up to 20% of the total DNA was conjugated to alpha 2-macroglobulin and the alpha 2-macroglobulin-DNA conjugate had a protein/DNA molar ratio of approximately two. The whole reaction takes place under very mild conditions in aqueous solution. The structure of DNA appears not to be significantly affected by the chemical modification. This method may prove useful in ligand directed gene transfer studies.
D003432:Cross-Linking Reagents; D003538:Cystamine / Q000031:analogs & derivatives; D004279:DNA, Viral / Q000378:metabolism*; D006801:Humans; D007096:Imidoesters; D008722:Methods; D010957:Plasmids; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics; D000511:alpha-Macroglobulins / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D007096:Imidoesters; D000511:alpha-Macroglobulins; C005905:methyl 4-mercaptobutyrimidate; C023874:N-acetyl-N'-(4-glyoxylylbenzoyl)cystamine; D013937:Thymidine Kinase; D003538:Cystamine