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.
Aoki
T
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Japan. aokit@hokuiryo-u.ac.jp
Tomaki
E
E
Satoh
M
M
Takashiro
M
M
Onagi
H
H
Itoh
M
M
Teramoto
T
T
Morikawa
J
J
Watabe
H
H
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