Condensed tannins are thought to benefit human health by serving as natural antioxidants, reportedly reducing cancer rates and heart disease. Anthocyanin reductase (ANR) was discovered to be a key enzyme controlling condensed tannin biosynthesis in dicot plants, but little has been published about the biochemical characterization of ANR enzymes from different species, and no antibodies are available. Our main emphasis was on trying to improve the expression levels of ANR protein in an E. coli pMAL expression system using an available Medicago truncatula ANR cDNA clone. NEB Express cells yielded more ANR-MBP protein (1.80 mg/0.25 L) than JM109 cells (1.05 mg/0.25 L). However, protein degradation was still evident, and expression of the full-length ANR appeared toxic to E. coli cells. Although full-length ANR-MBP protein fusions were observed on both dye-stained SDS-PAGE gels and Westerns using the anti-MBP antibodies, the accumulation levels were low compared to control proteins, and the ANR portion appears to be rapidly degraded, leaving mainly MBP protein. A quick cloning strategy allowed the removal of approximately 60% of the ANR protein, leaving the C-terminal portion fused to MBP. Much higher ANR-fragment-MBP protein accumulation was observed, although protein degradation during extraction and purification was still a problem. Currently the use of common protease inhibitors are being tested, to determine if these can inactivate the E. coli proteases to allow better recovery of intact fusion protein during Maltose/Amylose resin affinity chromatography. One of our long-term goals is to isolate sufficient purified ANR protein to generate high affinity antibodies for use in histology and future cloning of ANR from new species. In parallel, an attempt to isolate a new ANR cDNA clone from Cercis canadensis (redbud) seeds has been initiated. The roasted seeds were reported to be a component of diets of Native Americans, and it was previously demonstrated by colorimetric assays that the seeds are rich in condensed tannins. This project was supported by the National Institute of General Medical Sciences of the National Institutes of Health through Grant Number 8P20GM103447.and NASA Oklahoma Space Grant.

Expression in E. coli and purification of full-length or partial Medicago truncatula cDNA-encoded Anthocyanin Reductase (ANR) using MBP fusions

253rd National Meeting (2017)

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