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 T(0) 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.

Figure 1. Schematic representations of the T-DNA constructs used to express human C5a in tobacco; (A) vectors used for stable transformation targeting the recombinant protein to different subcellular compartments.

LB: left border; RB: right border; En: −340 bp to −91 bp CaMV 35S enhancer; p35S: −90 to −1 bp CaMV 35S core promoter; t35S: CaMV 35S terminator; Ω: 5′ Tobacco mosaic virus (TMV) untranslated region; nptII: neomycin phosphotransferase gene; ER: codon optimized signal peptide from the human IL6 gene; C5a: synthetic human C5a gene, codon optimized for tobacco; SEKDEL: ER retention motif; AFVY: vacuole-targeting peptide from common bean phaseolin protein; (B) Binary ‘MagnICON’ vectors used for transient expression. TVCV-Pol: RNA-dependent RNA polymerase from Turnip vein clearing virus (TVCV); MP: TMV movement protein; TVCV-3′-NTR: TVCV 3′ untranslated region; nos: A. tumefaciens nopaline synthetase gene terminator.

Figure 2. Protein expression levels in leaf extracts from T₀ transgenic plants determined using at least two ELISAs.

(A) Expression level measured in the individual transformants; Lane numbers indicate different independent tobacco events (NIV = wild-type control). (B) Box plot representation of C5a accumulation in the leaves of T₀ transformants following ANOVA (including the Bonferroni post-hoc test).

Figure 3. Average protein levels in the leaves and seeds of ten transgenic T₀ tobacco plants expressing different C5a variants, using TSP as the reference parameter, based on at least two independent ELISAs.

Figure 4. Protein expression levels in the T₁ generation of three independent transgenic tobacco lines accumulating C5a in the vacuole, as determined by at least two independent ELISAs.

(A) Expression level measured in the individual descendants; Lane numbers represent the different T₁ individuals and T₀ represents the parent. (B) Southern blots of the C5aVac 19 T₀ parent and selected T₁ and T₂ progeny (identified by lane numbers) and corresponding C5a expression levels determined by ELISA. The genomic DNA was digested with HindIII, which cuts once in the expression vector, and the presence of segregating bands indicates of three unlinked loci in the T₀ parent. M = DIG-labeled DNA Molecular Weight Marker II (Roche), NIV DNA from wild-type negative control plant.

Figure 5. Leaves of N. benthamiana Agroinfiltrated with either GFP-, C5aER- or C5aVac-expressing vectors pICH18711, pICH29912-C5aER and pICH29912-C5aVac, respectively.

One representative of at least three independent experiments is shown. (A) Pictures of leaves made under UV-light prior to sampling. dpi: days post inoculation (B) Yield of recombinant C5a as percentage of TSP in Agroinfiltrated leaves of N. benthamiana, sampled from 0–10 dpi. (C) Coomassie-stained SDS-PAGE of leaf extracts N. benthamiana leaves, Agroinfiltrated with GFP-expressing control vector pICH18711, sampled from 0–10 dpi.

Figure 6. Western Blot analysis of leaf and seeds samples of transgenic Geudertheimer plants (100 µg TSP); (A) Samples from stable transformed plants; E: 5 ng of recombinant C5a produced in E. coli; Control: leaf and seed samples from wild-type tobacco.

C5aVac: leaf and seed samples of the T₂ individual C5aVac 19-13-15, expressing the vacuolar variant of C5a; (B) Samples from N. benthamiana at 6 dpi after Agroinfiltration; control: N. benthamiana transfected with the empty vector pICH29912; C5aVac: N. benthamiana transfected with pICH29912-C5aVac; E: 5 ng of recombinant C5a produced in E. coli; (C) Verification of C5a biological activity using crude leaf extracts from transgenic tobacco and the near isogenic variant (wildtype) compared to a commercial standard C5a produced in E. coli. Enzyme release was measured by substrate conversion following exposure to different dilutions of the lead extracts, with the concentration of C5a determined by ELISA.