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Rivista di Biologia Molecolare e Biotecnologie

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Minerva Biotecnologica 2012 June;24(2):42-52

lingua: Inglese

Expression and purification of the light chain of human enteropeptidase in E. coli

Pepeliaev S. 1, Krahulec J. 2, Tlustá M. 1, Černý Z. 1, Jílková J. 1

1 CPN spol. s r.o., Dolní Dobrouč, Czech Republic;
2 Faculty of Natural Sciences, Comenius University in Bratislava, Department of Molecular Biology, Bratislava, Slovak Republic


Aim. The light chain of human enteropeptidase (enterokinase) is one of the most frequently used proteases because of its high specificity and activity. It cleaves proteins after the sequence (Asp)4Lys and leaves N-terminus of the downstream fused protein intact, it is stable in a broad range of pH and denaturing agent concentration. This work presents several attempts to produce the enterokinase in E. coli.
Methods. The enterokinase was expressed in a form of a single polypeptide chain as well as in fusion with the bacterial protein thioredoxin. In both cases the protein was expressed in form of inclusion bodies.
Results. The process of isolation and purification of enterokinase fused to thioredoxin is more effective in comparison with the fusion-free expressed enterokinase. The activity of the fusion-free enterokinase was about 17 U/mg while activity of the fused enterokinase was about 40 U/mg. Various methods of protein refolding were employed for enteokinase in vitro refolding.
Conclusion. It was found that the on-column refolding is more time- and cost-effective than dialysis and fast dilution methods.

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