Production of recombinant proteins through sequestration in chloroplasts: a strategy based on nuclear transformation and post-translational protein import

Abstract

Recently, plants have emerged as a lucrative alternative system for the production of recombinant proteins, as recombinant proteins produced in plants are safer and cheaper than those produced in bacteria and animal cell-based production systems. To obtain high yields in plants, recombinant proteins are produced in chloroplasts using different strategies. The first strategy is based on chloroplast transformation, followed by gene expression and translation in chloroplasts. This has proven to be a powerful approach for the production of proteins at high levels. The second approach is based on nuclear transformation, followed by post-translational import of proteins from the cytosol into chloroplasts. In the nuclear transformation approach, foreign genes are stably integrated into the nuclear genome or transiently expressed in the nucleus by non-integrating T-DNA. Although this approach also has great potential for protein production at high levels, it has not been thoroughly investigated. In this review, we focus on nuclear transformation-based protein expression and its subsequent sequestration in chloroplasts, and summarize the different strategies used for high-level production of recombinant proteins. We also discuss future directions for further improvements in protein production in chloroplasts through nuclear transformation-based gene expression.

Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

“…More recent work in N. benthamiana has demonstrated transient expression of both receptor binding and S1 domains of the SARS-CoV-2 spike protein and successful downstream application to immunoassays [19,20]. The main drawback of plant-based expression systems is that they have low biomass productivity compared to microbial systems and have laborious and technically demanding transformation methods that can require months to achieve if stable integration is required [21].…”

Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algaeChlamydomonas reinhardtii

“…The membrane (M) and nucleocapsid (N) proteins of SARS were expressed transiently in Nicotiana benthamiana, with the N protein demonstrating antibody recognition in convalescent serum samples (Demurtas et al, 2016). The main drawback of plant-based expression systems is that they have low biomass productivity compared to microbial systems, and have laborious and technically demanding transformation methods that can require months to achieve (Muthamilselvan et al, 2019).…”

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation

“…Third, plants are almost free of endotoxins such as lipopolysaccharides, which are abundant in bacterial cells. Further, the conditions for plant growth are much less affected by microorganisms that are detrimental to mammalian cell cultures (Buyel et al, 2017 ; Moon et al, 2019 ; Muthamilselvan et al, 2019 ; Knodler and Buyel, 2021 ; Schillberg and Finnern, 2021 ). In addition, previous studies indicated that various useful proteins of different origins, such as bacteria, bacteriophage, animals, and red algae, remained functional when produced in tobacco ( Nicotiana benthamiana ) (Garvey et al, 2013 ; Islam et al, 2019 , 2020 ; Kumari et al, 2020 ; Razzak et al, 2020 ).…”

“…Thus, to enhance the production of foreign proteins, several sequences such as 5′-untranslated region (UTR) and HSP transcriptional terminator have been formulated and incorporated into plant expression vectors (Nagaya et al, 2010 ; Kim et al, 2014 ; Islam et al, 2019 ). To avoid the proteolytic degradation of target proteins in the cytosol, expressed proteins can be sequestered to subcellular organelles such as endoplasmic reticulum (ER) or chloroplasts by incorporating organellar targeting signals that are proteolytically removed by organellar peptidase after organellar targeting of proteins (Lee and Hwang, 2018 ; Islam et al, 2019 ; Muthamilselvan et al, 2019 ; Margolin et al, 2020b ). ER is an organelle in which several posttranslational modifications such as glycosylation or disulfide bond formation occur, thereby contributing to the functionality of produced proteins (Margolin et al, 2018 , 2020a ).…”