Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

Abstract

We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T0 transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T2 generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.

Means to optimize protein expression in transgenic plants

“…Examples are the chloroplasts of N. benthamiana, A. thaliana and C. reinhardtii [63][64][65] as well as for example vacuole, ER and apoplast [66]. These examples demonstrate high gene expression levels and protein yields, underlining the importance of compartmentalization for improved protein expression.…”

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields

“…These C5a versions were also transiently expressed in Nicotiana benthamiana leaves using an hybrid binary vector (MagnICON) based on tobacco mosaic virus (TMV) that contains viral sequences required for RNA replication leading to amplification of RNA transcript, and also the highest yields were detected for vac-C5a variants (3.5-fold higher than ER). 28 Therefore vacuoles were found as the most suitable compartment to produce C5a, and the higher yields were attributed to the selection of the AFVY Ct, which is considered a PSV-specific targeting signal. 28 The authors argue that although lytic vacuoles are expected to be prevalent in vegetative tissues, the expression of storage protein derived sequence could induce the formation of storage organelles in vegetative tissue.…”

“…28 Therefore vacuoles were found as the most suitable compartment to produce C5a, and the higher yields were attributed to the selection of the AFVY Ct, which is considered a PSV-specific targeting signal. 28 The authors argue that although lytic vacuoles are expected to be prevalent in vegetative tissues, the expression of storage protein derived sequence could induce the formation of storage organelles in vegetative tissue. 28 Unexpected transient overexpression of ER-C5a and vac-C5a in Nicotiana benthamiana was accompanied by cytotoxic effects and a rapid decrease of recombinant C5a even though it is not anticipated that this protein could interfere with plant metabolism.…”

“…28 The authors argue that although lytic vacuoles are expected to be prevalent in vegetative tissues, the expression of storage protein derived sequence could induce the formation of storage organelles in vegetative tissue. 28 Unexpected transient overexpression of ER-C5a and vac-C5a in Nicotiana benthamiana was accompanied by cytotoxic effects and a rapid decrease of recombinant C5a even though it is not anticipated that this protein could interfere with plant metabolism. 28 Due to toxic effect, yields for vac-C5a (0,7%TSP) were higher than ER-C5a (0.2% TSP) but for non toxic protein using MagnICON system around 10% TSP were expected.…”

“…28 Unexpected transient overexpression of ER-C5a and vac-C5a in Nicotiana benthamiana was accompanied by cytotoxic effects and a rapid decrease of recombinant C5a even though it is not anticipated that this protein could interfere with plant metabolism. 28 Due to toxic effect, yields for vac-C5a (0,7%TSP) were higher than ER-C5a (0.2% TSP) but for non toxic protein using MagnICON system around 10% TSP were expected. Using the same sorting strategy, human interleukin (IL) 6 was expressed in stable transgenic N. tabacum and temporally in Nicotiana benthamiana, but ER sorted-IL6 produced yields 6.25 higher than vac-IL6, although IL6 was also fused to AFVY Ct VSS.…”