The first report on molecular cloning, functional expression, purification, and statistical optimization of Escherichia coli-derived recombinant Ficin from Iranian fig tree (Ficus carica cv.Sabz)

Abstract

The enzyme ficin, abundantly found in the leaves of the common Fig (Ficus carica. L), is a cysteine protease of the plant endopeptidase family. In terms of activity, this enzyme mimics the activity of the papain enzyme. However, the enzyme is more acidic than papain and binds with higher efficiency to its substrate. Ficin is widely used in the food and pharmaceutical industry along with the medical diagnosis. To date, there are no available data on cloning and recombinant production of various isoforms of ficin. In the present study, after the cloning process and optimized expression of ficin in E. coli BL21, by means of the central composite design (CCD) and approach-based response surface methodology (RSM), the recombinant protein was purified using the Ni-sepharose column and gel filtration. The activity of ficin was determined by its ability to hydrolyze the bovine casein enzyme as a substrate. These results showed the presence of different isoforms of ficin in this cultivar that they are distinct in terms of DNA coding sequences. The optimum conditions for maximum production of the recombinant ficin enzyme in E. coli were as follows; a cell density of 1.25, post-induction time 7 h, 10% (w/v) lactose concentration, and shaking at 115 rpm at 24 °C. The concentration of purified product was reported to be 0.27 mg/ml. The optimization procedures increased the amounts of ficin production by approximately 3 folds (0.67 mg/ml) compared with the expiration level (in the absence of optimization). Also, our findings showed that the recombinant ficin was able to hydrolyze casein, denoting the functionality of the enzyme when used in-vitro. The pitfall of cutting-off the young branches of the common fig tree to purify the enzyme from the young shoots was successfully solved in this study.