Cloning, Expression, and Purification Strategies for Enhanced Production of Enterokinase using TrpE fusion tag in Bench Scale Bioreactor

Publish Year: 1400
نوع سند: مقاله ژورنالی
زبان: English
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تاریخ نمایه سازی: 15 دی 1400


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 TrpLE۱۴۱۳ (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 BL۲۱ (DE۳) 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 ۲۴۱ mg/L of recombinant EK, and its purity confirmed by RP-HPLC was greater than ۹۷%. However, the maximum EK yield reported by other researchers is only ۱۰۶ 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 ۲-۲.۵ 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.


Santhosh Nagaraj Nanjundaiah

Discovery Biology, Anthem Biosciences Pvt Ltd, Bommasandra, Bangalore, India

Jayasri MA

School of Biosciences and Technology, Vellore Institute of Technology, Vellore, India

Sunilkumar Sukumaran

Discovery Biology, Anthem Biosciences Pvt Ltd, Bommasandra, Bangalore, India

Ganesh Sambasivam

Discovery Biology, Anthem Biosciences Pvt Ltd, Bommasandra, Bangalore, India