Journal
PHYTOCHEMICAL ANALYSIS
Volume 34, Issue 6, Pages 632-640Publisher
WILEY
DOI: 10.1002/pca.3251
Keywords
deoxymiroestrol; Escherichia coli; extraction; fragment antigen-binding antibody; miroestrol; Pueraria mirifica
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Two immunoaffinity separation approaches using active fragment antigen-binding (Fab) antibodies were developed to selectively separate miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica. The methods were successful in purifying the compounds from the plant extract.
IntroductionMiroestrol 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 (R) 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 mu g 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 mu g 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.
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