Expression of blood clot‐dissolving proteins in transgenic plant (LB852)

Abstract

Introduction: The formation of a blood clot in the circulatory system can cause heart attack or stroke. Lumbrokinases, found in earthworms, and Nattokinase, found in Bacillus subtilis for soybean fermentation, can dissolve fibrin clots without causing excessive bleeding. Our goal was to clone Lumbrokinase and Nattokinase, to transiently and stably express the proteins in tobacco plants and to prove these recombinant proteins retain anti‐thrombolytic activity.Methods: Codon‐optimized and wild‐type gene fragment of Lumbrokinases (PI293) and Nattokinase (GenBank: AF368283.1) were isolated and inserted into a single‐vector DNA replicon system for transient expression in plant and plant expression vector pCambia2300‐Phas1470‐Nos, for stable plant transformation and expression. We tested gene expression and function of the transient expression vector using fibrin dissolving assay.Results: We optimized Lumbrokinase and Nattokinase gene codon respectively for tobacco codon usage bias. Upon codon optimization, codon adaptation index (CAI) of Lumbrokinase was increased from 0.69 to 0.86 and GC content was changed from 54.02% to 42.65%. CAI of Nattokinase was increased from 0.71 to 0.86 and GC content was changed from 46.78% to 42.61%. Higher CAI value and GC content, closer to that of tobacco plant, provide a good estimation for high expression foreign protein. Total eight vectors for transient expression and stable transformation were developed and confirmed by restriction reactions. The plant transient expression with those vectors tested positive for gene expression and function with the fibrin dissolving assay.Conclusions: Expression Vectors of Lumbrokinase and Nattokinase for both transient and stable plant transformation were developed. The transient expression vectors were successfully expressed in the tobacco plant and tested positive for clot dissolving function.Grant Funding Source: National Institutes of Health through Grant Number 8P20GM103447