Approaches to achieve high‐level heterologous protein production in plants

Abstract

SummaryPlants offer an alternative to microbial fermentation and animal cell cultures for the production of recombinant proteins. For protein pharmaceuticals, plant systems are inherently safer than native and even recombinant animal sources. In addition, post-translational modifications, such as glycosylation, which cannot be achieved with bacterial fermentation, can be accomplished using plants. The main advantage foreseen for plant systems is reduced production costs. Plants should have a particular advantage for proteins produced in bulk, such as industrial enzymes, for which product pricing is low. In addition, edible plant tissues are well suited to the expression of vaccine antigens and pharmaceuticals for oral delivery. Three approaches have been followed to express recombinant proteins in plants: expression from the plant nuclear genome; expression from the plastid genome; and expression from plant tissues carrying recombinant plant viral sequences. The most important factor in moving plant-produced heterologous proteins from developmental research to commercial products is to ensure competitive production costs, and the best way to achieve this is to boost expression.Thus, considerable research effort has been made to increase the amount of recombinant protein produced in plants. This research includes molecular technologies to increase replication, to boost transcription, to direct transcription in tissues suited for protein accumulation, to stabilize transcripts, to optimize translation, to target proteins to subcellular locations optimal for their accumulation, and to engineer proteins to stabilize them. Other methods include plant breeding to increase transgene copy number and to utilize germplasm suited to protein accumulation. Large-scale commercialization of plant-produced recombinant proteins will require a combination of these technologies.

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

“…The various factors that affect the expression of a particular heterologous gene and subsequent production of its protein in plant cells include: (i) transcriptional determinants such as transgene copy number, site of integration of the T-DNA in the chromosome, and promoter activity [50]; (ii) posttranscriptional factors including, 5 0 -untranslated leader sequence, 3 0 -polyadenylation, and mRNA stability [51,52]; and (iii) translational/post-translational factors such as protein stability, modification, and trafficking [52,53]. Promoters and enhancers (cis-acting regulatory elements) play a crucial role in regulating spatial and temporal patterns of transgene expression at particular stages or in response to particular conditions [50][51][52][53][54][55].…”

“…Promoters and enhancers (cis-acting regulatory elements) play a crucial role in regulating spatial and temporal patterns of transgene expression at particular stages or in response to particular conditions [50][51][52][53][54][55]. Several promoters and other cis-acting elements including signal peptides of plant, plant pathogen, or bacterial origin used in heterologous expression vectors have been described (Table S1 in the supplementary material online).…”

“…The most obvious is the relatively low level of in situ protein expression/accumulation observed in most studies (Table S1 in the supplementary material online), in which an order-of-magnitude increase in expression level is required for complete conversion of biomass [8,33]. In addition to the obvious requirement for careful codon optimization [8,46,59] and/or codon harmonization [69], the further development of combinations of transit peptides and promoters (strong promoters and chimeric promoters), with or without promoter engineering, may be effective strategies [52,70]. Carbohydrate-binding modules can also be used to target better the recombinant enzymes and ultimately improve biomass digestion efficiency [45].…”

“…Transgenic lines containing multiple transgene copies from different events appear to have a reduced risk of gene silencing, compared to those engineered by multiple gene insertions during the same transformation events [12,52].…”

Investigating recombinant protein exudation from roots of transgenic tobacco

“…Plants, seeds and cultured plant cells are potentially one of the most economical systems for large-scale production of recombinant proteins for the biochemical, veterinary and pharmaceutical industries (Boehm, 2007;Floss et al, 2007;Kermode, 2006;Pujol et al, 2007;Streatfield, 2007;Twyman et al, 2005). The well-established current state of plant molecular farming is in fact contributing to the extensive development and wide acceptance of this attractive and powerful technology.…”