Immunoaffinity separation of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham using fragment antigen‐binding antibody produced via Escherichia coli

Abstract

AbstractIntroductionMiroestrol and deoxymiroestrol are potent phytoestrogens and are oestrogen markers of Pueraria candollei var. mirifica. However, purifying these compounds is difficult because they only exist in trace amounts.ObjectivesActive fragment antigen‐binding (Fab) antibodies were produced via Escherichia coli SHuffle® T7 and used to selectively separate these compounds.Materials and methodsTwo immunoaffinity separation approaches were developed, namely the immunoaffinity column (IAC) and a cell‐based method. Group‐specific Fab antibodies against miroestrol and deoxymiroestrol (anti‐MD Fab) were used as biological binding reagents for selective separation.ResultsThe Fab‐based IAC effectively separated miroestrol and deoxymiroestrol (0.65 and 2.24 μg per 2 mL of resin, respectively) from P. mirifica root extract. When P. mirifica extract was added to E. coli cultures during Fab expression via a cell‐based method, the target compound accumulated in intracellular compartments and, thus, were separated from E. coli cells after the removal of other compounds. A yield of 1.07 μg of miroestrol per gram of cell pellet weight was obtained. Miroestrol and deoxymiroestrol were successfully purified from P. mirifica extract using anti‐MD Fab via the IAC and an intracellular cell‐based method.ConclusionThe proposed methods can simplify the miroestrol and deoxymiroestrol extraction process and provide a basis for applications utilising recombinant antibodies to separate target compounds.