Bovine pancreatic trypsin inhibitor-trypsin complex as a detection system for recombinant proteins.
Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10(-14) and 1.1 x 10(-13) M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.
Borjigin
J
J
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Nathans
J
J
D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D006801:Humans; D007202:Indicators and Reagents; D017433:Protein Structure, Secondary; D011817:Rabbits; D011986:Receptors, Somatotropin / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000378:metabolism; D017299:Rod Opsins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D013053:Spectrophotometry; D014162:Transfection; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D007202:Indicators and Reagents; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; D007611:Aprotinin; D014357:Trypsin