Reversible immunoprecipitation using histidine- or glutathione S-transferase-tagged staphylococcal protein A.
We have constructed, expressed, and purified hexahistidine- and glutathione S-transferase (GST)-tagged Staphylococcal protein A. The histidine-tagged protein A bound efficiently to iminodiacetic acid (IDA)-Sepharose loaded with Zn2+, and the GST-protein A was efficiently retained by glutathione-Sepharose. Both recombinant forms of protein A can be used in the normal way to harvest immune complexes with IgG. Both forms of protein A can be released from the Sepharose matrix by mild procedures. The his6-protein A:antibody:antigen complexes can be released from the matrix with EDTA, and immunoprecipitates bound to GST-protein A can be released either by elution with glutathione or by digestion with thrombin. We tested this method with immunoprecipitates of the p40MO15 protein kinase, and found that they retained their ability to phosphorylate p33cdk2 after elution from the affinity matrices.
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Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar, Hertfordshire, United Kingdom.
Hunt
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D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D005815:Genetic Code; D005982:Glutathione Transferase / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose; D013205:Staphylococcal Protein A / Q000737:chemistry*
D003062:Codon; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D006639:Histidine; D012685:Sepharose; D005982:Glutathione Transferase