Detection of blotted antigen using a fusion protein between protein A and pepsinogen C.
Human pepsinogen (PG) A and C were fused with protein A and expressed in Escherichia coli. Although the fusion proteins (PA-PGA and PA-PGC) were not expressed at high levels and were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. PA-PGA and PA-PGC possessed proteolytic activity equivalent to the gastric mucosal PGA and PGC, respectively. However, the activity of PA-PGC was about 3-fold higher than that of PA-PGA. Therefore, PA-PGC was applied to the subsequent immunoblotting studies. The proteolytic activity of PA-PGC was used for digesting the blocking reagent around the target antigen (in situ digestion method) or casein-clotting in the agarose plate containing skimmed milk (caseogram print method). Although the sensitivity of these methods was lower than that of the conventional color detection, the caseogram print method was superior in that the reaction was linear over a wide range. On the other hand, the in situ digestion method possessed a unique property on Western blotting, and it was very easy to identify the relative position of the target, which could be recognized as a clear band. For PA-PGC detection, no special chemicals are required, and so the procedure is simple, rapid, and inexpensive.
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, 061-0293, Japan. aokit@hoku-iryo-u.ac.jp
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D000941:Antigens / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020115:Pepsinogen C / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000378:metabolism*
D000941:Antigens; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D020115:Pepsinogen C