Purification of recombinant human pepsinogens and their application as immunoassay standards.
Human pepsinogen (PG) A and C were cloned in Escherichia coli, but the levels of expression were low and unstable. When there were fused to maltose-binding protein (MBP), the fusion proteins (MBP-PGA and MBP-PGC) were expressed as the major products. Although these fused products were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. MBP-PGA and the PGA segment obtained by factor Xa digestion (designated as r-PGA) possessed proteolytic activities equivalent to native PGA purified from gastric tissue (t-PGA). For PGCs (MBP-PGC, r-PGC and t-PGC) also, the specific activities were almost the same. However, the activities of PGCs were about 3- to 4-hold higher than those of PGAs. In PGA and PGC immunoassay systems, r-PGs (r-PGA and r-PGC) and the EIA kit standard PGs (gastric mucosal PGs) exhibited a good correlation. From these results, r-PGs would seem to be applicable as assay standards without compromising the sensitivity of the immunoassay systems.
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Japan. aokit@hokuiryo-u.ac.jp
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T
Aoki
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Tomaki
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Satoh
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Takashiro
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Onagi
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Itoh
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Teramoto
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Morikawa
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Watabe
D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002352:Carrier Proteins; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay / Q000379:methods*; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010435:Pepsinogens / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012015:Reference Standards
D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010435:Pepsinogens; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli