Transient protein expression in tobacco BY-2 plant cell packs using single and multi-cassette replicating vectors

Abstract

Key message This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Abstract Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.

Transient recombinant expression of highly immunogenic CagA, VacA and NapA in Nicotiana benthamiana

“…However, the high transcript levels of CagA, VacA and NapA did not completely correspond to the protein yields, which could be because that the quantity of mRNA levels does not restrict translation and that post-translation modification can lead to the lysis of proteins [56] . A similar result was reported in a recent study [57] , where the higher transcript levels of green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRed) transiently expressed in tobacco BY-2 plant cell packs did not necessarily correspond to the protein yield.…”

Extended Set of GoldenBraid Compatible Vectors for Fast Assembly of Multigenic Constructs and Their Use to Create Geminiviral Expression Vectors

“…Based on our results the TU positioned nearest to the left border is expressed more strongly than TU located downstream ( Figure 5). Recently we have also obtained similar results from plant cell packs derived from tobacco BY-2 cells (Poborilova et al, 2020). Co-ordinated co-expression of two or more transcripts from a single vector is important for the expression of multisubunit proteins such as virus like particles, antibodies or vaccines.…”

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana

“…Transient expression systems have surpassed the challenge of rapid and high-yield expression of recombinant proteins in plants, allowing for gram-sized quantities to be attained in as little as 5 days [7,8]. However, so far only a few examples of the use of a transient expression in suspension cell cultures are known in the literature [9,10]. One of the reasons for the low yield of recombinant protein is the insertion of the transgene into a random genome region, which leads to different levels of expression due to the positional effects and different numbers of transgene copies.…”