Plant-Made Bet v 1 for Molecular Diagnosis

Abstract

Allergic disease diagnosis is currently experiencing a breakthrough due to the use of allergenic molecules in serum-based assays rather than allergen extracts in skin tests. The former methodology is considered a very innovative technology compared with the latter, since it is characterized by flexibility and adaptability to the patient's clinical history and to microtechnology, allowing multiplex analysis. Molecular-based analysis requires pure allergens to detect IgE sensitization, and a major goal, to maintain the diagnosis cost-effective, is to limit their production costs. In addition, for the production of recombinant eukaryotic proteins similar to natural ones, plant-based protein production is preferred to bacterial-based systems due to its ability to perform most of the post-translational modifications of eukaryotic molecules. In this framework, Plant Molecular Farming (PMF) may be useful, being a production platform able to produce complex recombinant proteins in short time-frames at low cost. As a proof of concept, PMF has been exploited for the production of Bet v 1a, a major allergen associated with birch (Betula verrucosa) pollen allergy. Bet v 1a has been produced using two different transient expression systems in Nicotiana benthamiana plants, purified and used in a new generation multiplex allergy diagnosis system, the patient-Friendly Allergen nano-BEad Array (FABER). Plant-made Bet v 1a is immunoreactive, binding IgE and inhibiting IgE-binding to the Escherichia coli expressed allergen currently available in the FABER test, thus suggesting an overall similar though non-overlapping immune activity compared with the E. coli expressed form.

Keywords: IgE; allergen; molecular farming; structure homology; transient expression.

Figure 1

Time-course analysis of pK7WG2. Betv1 expression of the agro-infiltrated N. benthamiana leaves. Western blot analysis (A) and corresponding loading controls (RuBisCO large subunit) stained with Coomassie Brilliant Blue (B), of pBet v 1a containing protein extract from leaves samples collected from 3 to 14 dpi. Each lane was loaded with 2.5 µg of TSP, the western blot was probed with anti-FLAG® antibody conjugated with horseradish peroxidase. Side numbers indicate molecular mass markers in kDa. p.c., positive control, 10 ng of a commercially available flagged protein; n.c., negative control, extract from leaves infiltrated solely with A. tumefaciens EHA105.

Figure 2

Evaluation of extraction reproducibilty of the pG PVX GATEWAY(A).Betv1 agro-infiltrated N. benthamiana leaves. Western blot analysis (A) and corresponding loading controls (RuBisCO large subunit) stained with Coomassie Brilliant Blue (B), of pBet v 1a containing protein extract from leaves samples in three biological replicates. Each lane was loaded with 2.5 µg of TSP, the western blot was probed with anti-FLAG® antibody conjugated with horseradish peroxidase. Side numbers indicate molecular mass markers in kDa. p.c., positive control, 10 ng of acommercially available flagged protein; n.c., negative control, extract from leaves infiltrated solely with A. tumefaciens GV3101.

Figure 3

Evaluation of the pBet v 1a Purification, from pK7WG2. Betv1 agroinfected leaves, using a imidazole gradient. Western blot analysis (A) and corresponding Silver Staining (B), of 1.5 mL purification elutions. Each lane was loadedwith 5 µL of elution, the western blot was probed with anti-FLAG® antibody conjugated with horseradish peroxidase. Side numbers indicate molecular mass markers in kDa. p.c., positive control, 10 ng of a commercially available flagged protein; n.c., negative control, from leaves infiltrated solely with A. tumefaciens EHA105, submitted to the same purification protocol as the leaves extract containing pBet v 1a. The bottom bar of the B image stands for the imidazole concentration for every gradient fraction.

Figure 4

Average yields µg/g LFW of purified protein between the two expression systems. The expression and purification yields are reported, respectively, in green and yellow. The bars represent the standard deviations of three independent purification experiments, asterisks indicates the statiscal significance evaluated by t-test (p < 0.01).

Figure 5

Gel filtration profile on a Superdex 75 column of purified pBet v 1a and Bet v 1 expressed in E. coli.

Figure 6

RP-HPLC elution profile of purified pBet v 1a (200 µg).

Figure 7

Multiple-sequence alignment of Bet v 1a (accession number P15494) with the homologous allergens Cor a 1, Mal d 1, Ara h 8, Api g 1 and Act c 11 having the accession number Q08407, 598 Q9SYW3, Q6VT83, P49372, A0A2R6PAW0, respectively. The residues of Bet v 1a conserved in the aligned homologues are shadowed in gray. The amino acid residues experimentally identified by mass spectrometry are in white and black shadows.

Figure 8

CD spectra of plant-expressed and E. coli–expressed (commercial) Bet v 1.

Figure 9

Molar ellipticity of pBet v 1a at 222 nm at the analyzed temperatures.

Figure 10

(A) Specific IgE-binding inhibition using pBet v 1a on Bet v 1-like molecules on the FABER test solid phase. (B) Specific IgE direct comparative binding using 304 Bet v 1 IgE-positive sera and statistical evaluation of IgE value distributions by using the t-test for paired values (p < 0.0001), displayed by the asterisk.

Figure 11

Streamline pBet v 1a production and characterization.