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

Introduction: Miroestrol 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.

Objectives: Active fragment antigen-binding (Fab) antibodies were produced via Escherichia coli SHuffle® T7 and used to selectively separate these compounds.

Materials And Methods: Two 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.

Results: The 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.

Conclusion: The proposed methods can simplify the miroestrol and deoxymiroestrol extraction process and provide a basis for applications utilising recombinant antibodies to separate target compounds.