Introduction: Enterokinase (EK) is an enzyme of the serine protease family which is widely used in protein purification. EK acts at the C terminus of the DDDDK site in a protein chain. The enzyme has gained commercial importance in recent times owing to its specificity in biosimilar processing and also while removing the fusion tags in the course of protein purification.
Materials and Methods: The commercial production of EK has faced several challenges and demands the usage of novel strategies. This research shows the construction of a vector using TrpLE1413 (TrpE) as a fusion tag that pushes the produced EK inside the cell towards the inclusion body fraction and produced more of the desired protein in the BL21 (DE3) strain of Escherichia coli. The inclusion bodies produced by fed-batch fermentation were solubilized, refolded, activated, and purified by a single step of anion exchange chromatography.
Results: We purified 241 mg/L of recombinant EK, and its purity confirmed by RP-HPLC was greater than 97%. However, the maximum EK yield reported by other researchers is only 106 mg/L.
Conclusions: Overall, our results demonstrate the potential of the TrpE fusion tag along with novel expression and purification strategies to increase the enzyme yield by 2-2.5 times when compared to the yield achieved using traditional methods. Hence, this study has paved the way for the industrial production of EK in an economically viable manner. The same strategy could possibly be implemented on the expression of other industrially important recombinant enzymes depending on the protein characteristics.