Production of Bioactive Recombinant Reteplase by Virus-Based Transient Expression System in Nicotiana benthamiana

Abstract

To explore a cost-effective alternative method to produce the recombinant thrombolytic drug Reteplase (rPA), a plant viral amplicon-based gene expression system was employed to transiently express bioactive Strep II-tagged recombinant rPA in Nicotiana benthamiana leaves via agro-infiltration. Several gene expression cassettes were designed, synthesized in vitro, and then cloned into Tobacco mosaic virus RNA-based overexpression vector. Codon optimization, subcellular targeting, and the effect of attached Strep-tag II were assessed to identify conditions that maximized expression levels of the recombinant rPA in tobacco leaves. We found that codon-optimized rPA with N-terminal Strep-tag II that was aimed to the endoplasmic reticulum as target provided the highest amount of biologically active protein, i.e., up to ∼50 mg from per kilogram fresh weight leaf biomass in less than 1 week. Furthermore, the recombinant rPA was conveniently purified from inoculated leaf extracts by a one-step purification procedure via the Strep-tag II. The plant-made rPA was glycosylated with molecular mass of ∼45.0 kDa, and its in vitro fibrinolysis activity was equivalent to the commercial available rPA. These results indicate that the plant viral amplicon-based system offers a simple and highly effective approach for cost-effective large-scale production of recombinant rPA.

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo

“…The 72- and 40-kDa bands are also detected with anti-tPA antibodies but the 34-kDa band reacts only to anti-IgG antibodies ( Fig 4B ). Protein cleavage was also observed for tPA [ 33 , 58 ] and rPA expressed in Nicotiana species [ 34 ]. In general, tPA and rPA expressed in eukaryote cells can be hydrolysed (R-I bond in K2, S1 Fig ) into A and B-chains by plasmin, trypsin and other serine proteases [ 33 , 58 ].…”

“…Similar results were observed when strep-tagged rPA was expressed in N . benthamiana and degradation of the 45-kDa protein to 31- and 14-kDa products was only visible when the sample was analysed under reducing conditions [ 34 ].…”

“…Reteplase has been expressed in other hosts such as Bacillus [ 30 ]. Yeast [ 31 ]., insect [ 32 ], mammalian [ 23 , 24 ] and plant cells [ 33 , 34 ]. The expression levels and protein stability vary significantly among all these systems.…”

Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

“…The expression of T-DNA carrying the gene of interest occur in either transient or stable manner was demonstrated by Krenek et al [ 30 ], Janssen and Gardner [ 95 ]. Both stable and transient transformation methods can be utilized in genome editing [ 96 ], to study the gene and protein function [ 97 , 98 ], molecular processes [ 99 ] and are applicable to numerous plant species [ 100 ]. Heenatigala et al [ 99 ] have developed efficient protocols for stable and transient gene transformation for Wolffia globosa using Agrobacterium that has application in a wide range of scientific and commercial processes.…”

Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant

“…In general, there are two approaches to express recombinant proteins in plants, which are transgenic and transient plant expression ( Chen et al, 2013 ; Joh, McDonald & VanderGheynst, 2006 ; Ma, Li & Wang, 2019 ; Wydro, Kozubek & Lehmann, 2006 ). For transient expression, the vector pEAQ- HT , which is based on the Cowpea mosaic virus (CPMV), has been reported to be an efficient expression vector for therapeutic recombinant proteins ( Shah, Almaghrabi & Bohlmann, 2013 ).…”