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Evidence-Based Complementary and Alternative Medicine
Volume 2017, Article ID 6093017, 9 pages
Research Article

Transient Expression of Lumbrokinase (PI239) in Tobacco (Nicotiana tabacum) Using a Geminivirus-Based Single Replicon System Dissolves Fibrin and Blood Clots

1Department of Natural Sciences, Northeastern State University, Broken Arrow, OK, USA
2Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China
3Laboratory of Comparative Neuroimmunology, Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
4Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA

Correspondence should be addressed to Dehu Liu; moc.621@6002uheduil and Kevin Yueju Wang; ude.kousn@30gnaw

Received 6 February 2017; Revised 18 June 2017; Accepted 17 July 2017; Published 28 August 2017

Academic Editor: Gioacchino Calapai

Copyright © 2017 Alexia Dickey et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild-type (WT) and plant codon-optimized (OP) PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum) leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.