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

Abstract

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.

The potential role of ischaemia–reperfusion injury in chronic, relapsing diseases such as rheumatoid arthritis, Long COVID, and ME/CFS: evidence, mechanisms, and therapeutic implications

“…Serrapeptase (serratiopeptidase) [ 934 , 956–959 ] has a similar activity (as well as others such as mucolytic behaviour [ 958 ]) and comes from a Serratia marcescens strain that originates in the guts of silkworms, where it has a natural role in helping the worms emerge from their cocoons. Lumbrokinases are another orally active [ 960 ] set of fibrinolytic enzymes that have been found in earthworms [ 961–965 ], and may also be produced recombinantly [ 966 , 967 ]; they may also have some tPA activity [ 965 ]. Each has been proposed as of value in acute and/or Long COVID treatment [ 751 , 958 , 968 ].…”

Uji Aktivitas Fibrinolitik Jus Cacing Tanah (Lumbricus rubellus) Pada Tikus yang Diberi Asam Traneksamat

“…Selain karena faktor lamanya waktu perlakuan, hal ini diduga karena target aktivitas enzim lumbrokinase secara khusus pada fibrin dan tidak spesifik pada penurunan trombosit. Sementara agen trombolitik lainnya seperti tPA, streptokinase, dan urokinase (uPA) tidak secara khusus ditargetkan untuk fibrin (Dickey et al 2017). Enzim lumbrokinase membantu mengurangi agregasi trombosit melalui rantai reaksi pada fibrin, cAMP, dan kalsium, yang dikaitkan dengan kenaikan waktu perdarahan dan waktu pembekuan (Sukmawan, 2020;Ji et al 2008).…”

Application of Plant Viruses in Biotechnology, Medicine, and Human Health

“…Bean yellow dwarf virus (BeYDV) is a geminivirus frequently used for expression of pharmaceutical proteins. BeYDV has recently been used to produce norovirus, HIV, HPV, and hepatitis B virus subunit vaccines, monoclonal antibodies to West Nile virus and Ebola virus, as well as earthworm-derived Lumbrokinase (PI239), used to dissolve fibrin and blood clots [ 149 , 150 ]. Besides using higher plants such as tobacco as hosts, geminiviruses have also been used to express proteins in algae [ 151 ].…”