Michael D. McLean

Metabolism and functions of gamma-aminobutyric acid

Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. In plants, stress initiates a signal-transduction pathway, in which increased cytosolic Ca2+ activates Ca2+/calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. Elevated H+ and substrate levels can also stimulate glutamate decarboxylase activity. GABA accumulation probably is mediated primarily by glutamate decarboxylase. However, more information is needed concerning the control of the catabolic mitochondrial enzymes (GABA transaminase and succinic semialdehyde dehydrogenase) and the intracellular and intercellular transport of GABA. Experimental evidence supports the involvement of GABA synthesis in pH regulation, nitrogen storage, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization. There is a need to identify the genes of enzymes involved in GABA metabolism, and to generate mutants with which to elucidate the physiological function(s) of GABA in plants.

Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol.

A single-domain variable heavy chain (V(H)H) antibody fragment specific to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a llama (Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax Classic adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional (cIgG) and heavy chain antibody (HCAb) fractions, a V(H)H library was constructed from amplified cDNA by nested PCR. V(H)H fragments with binding affinity for the mycotoxin were selected by panning of the phagemid library against microtiter plates coated with 15-DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified as a V(H)H monomer (mNAT-267) at a final concentration of 1.3 mg mL(-1). Isolated NAT-267 V(H)H DNA was fused to the homopentamerization domain of the B subunit of verotoxin to generate the pentabody format of single-domain antibody (sAb). The V(H)H pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration of 1.0 mg mL(-1). Surface plasmon resonance (SPR) analysis of soluble mNAT-267 binding kinetics to immobilized 15-DON-Horse Radish Peroxidase (HRP) indicated a dissociation constant (K(D)) of 5microM. Competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and fluorescence polarization assay (FPA) inhibition experiments with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition FPAs with mNAT-267 and pNAT-267 determined IC(50) values of 1.24 and 0.50 microM, respectively, for 15-AcDON hapten. These values were similar to the IC(50) value of 1.42 microM for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization. Competition formats for structurally related trichothecenes resulted in no cross-reactivity to: DON; 3-acetyldeoxynivalenol (3-AcDON); neosolaniol (NEO); diacetoxyscirpenol (DAS); and T-2 toxin. Our study confirmed that recombinant V(H)H fragments capable of binding low molecular weight haptens can be produced through the creation and panning of hyper-immunized single-domain (sdAb) libraries.

Overexpression of glutamate decarboxylase in transgenic tobacco plants confers resistance to the northern root-knot nematode

Previous research suggests that the endogenous synthesis of gamma-aminobutyrate (GABA), a naturally occurring inhibitory neurotransmitter, serves as a plant defense mechanism against invertebrate pests. Here, we tested the hypothesis that elevated GABA levels in engineered tobacco confer resistance to the northern root nematode (Meloidogyne hapla). This nematode species was chosen because of its sedentary nature and economic importance in Canada. We derived nine phenotypically normal, homozygous lines of transgenic tobacco (Nicotiana tabacum L.), which contain one or two copies of a full-length, chimeric tobacco glutamate decarboxylase (GAD) cDNA or a mutant version that lacks the autoinhibitory calmodulin-binding domain, under the control of a chimeric octopine synthase/mannopine synthase promoter. Regardless of experimental protocol, uninfected transgenic lines consistently contained higher GABA concentrations than wild-type controls. Growth chamber trials revealed that 9–12 weeks after inoculation of tobacco transplants with the northern root-knot nematode, mature plants of five lines possessed significantly fewer egg masses on the root surface when the data were expressed on both root and root fresh weight bases. Therefore, it can be concluded that constitutive transgenic expression of GAD conferred resistance against the root-knot nematode in phenotypically normal tobacco plants, probably via a GABA-based mechanism.

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.

Inheritance of picloram and 2,4-D resistance in wild mustard (Brassica kaber)

Abstract The primary goal of this research was to determine the inheritance of cross-resistance to several groups of auxinic herbicides through classical genetic approaches using auxinic herbicide–resistant (R) and –susceptible (S) wild mustard biotypes obtained from western Canada. F1 progeny were raised from crosses between homozygous auxinic herbicide–R and –S wild mustard parental lines. The F1 and F2 populations were assessed for picloram (pyridine group) and 2,4-D (phenoxyalkanoic group) resistance or susceptibility. Analyses of the F1 as well as the F2 progeny indicate that a single dominant gene confers the resistance to picloram and 2,4-D similar to an earlier report of dicamba-based (benzoic acid group) resistance in this wild mustard biotype. Furthermore, analyses of backcross progeny in this species indicate that resistance to all three auxinic herbicides, i.e., picloram, dicamba, and 2,4-D, is determined by closely linked genetic loci. With this information on inheritance of resistance to several auxinic herbicide families, the R biotype of wild mustard offers an excellent system to isolate and characterize the auxinic herbicide–resistance gene. Nomenclature: 2,4-D; picloram; wild mustard, Brassica kaber (DC) L.C. Wheeler SINAR.

Purification of a human immunoglobulin G1 monoclonal antibody from transgenic tobacco using membrane chromatographic processes.

Efficient purification of protein biopharmaceuticals from transgenic plants is a major challenge, primarily due to low target protein expression levels, and high impurity content in the feed streams. These challenges may be addressed by using membrane chromatography. This paper discusses the use of cation-exchange and Protein A affinity-based membrane chromatographic techniques, singly and in combination for the purification of an anti-Pseudomonas aerugenosa O6ad human IgG1 monoclonal antibody from transgenic tobacco. Protein A membrane chromatography on its own was unable to provide a pure product, mainly due to extensive non-specific binding of impurities. Moreover, the Protein A membrane showed severe fouling tendency and generated high back-pressure. With cation-exchange membrane chromatography, minimal membrane fouling and high permeability were observed but high purity could not be achieved using one-step. Therefore, by using a combination of the cation-exchange and Protein A membrane chromatography, in that order, both high purity and recovery were achieved with high permeability. The antibody purification method was first systematically optimized using a simulated feed solution. Anti-P. aeruginosa human IgG1 type monoclonal antibody was then purified from transgenic tobacco juice using this optimized method.

PLANT-PRODUCED TRASTUZUMAB INHIBITS THE GROWTH OF HER2 POSITIVE CANCER CELLS

To study the agricultural production of biosimilar antibodies, trastuzumab (Herceptin) was expressed in Nicotiana benthamiana using the magnICON viral-based transient expression system. Immunoblot analyses of crude plant extracts revealed that trastuzumab accumulates within plants mostly in the fully assembled tetrameric form. Purification of trastuzumab from N. benthamiana was achieved using a scheme that combined ammonium sulfate precipitation with affinity chromatography. Following purification, the specificity of the plant-produced trastuzumab for the HER2 receptor was compared with Herceptin and confirmed by western immunoblot. Functional assays revealed that plant-produced trastuzumab and Herceptin have similar in vitro antiproliferative effects on breast cancer cells that overexpress HER2. Results confirm that plants may be developed as an alternative to traditional antibody expression systems for the production of therapeutic mAbs.

Plant-based solutions for veterinary immunotherapeutics and prophylactics

An alarming increase in emergence of antibiotic resistance among pathogens worldwide has become a serious threat to our ability to treat infectious diseases according to the World Health Organization. Extensive use of antibiotics by livestock producers promotes the spread of new resistant strains, some of zoonotic concern, which increases food-borne illness in humans and causes significant economic burden on healthcare systems. Furthermore, consumer preferences for meat/poultry/fish produced without the use of antibiotics shape today’s market demand. So, it is viewed as inevitable by the One Health Initiative that humans need to reduce the use of antibiotics and turn to alternative, improved means to control disease: vaccination and prophylactics. Besides the intense research focused on novel therapeutic molecules, both these strategies rely heavily on the availability of cost-effective, efficient and scalable production platforms which will allow large-volume manufacturing for vaccines, antibodies and other biopharmaceuticals. Within this context, plant-based platforms for production of recombinant therapeutic proteins offer significant advantages over conventional expression systems, including lack of animal pathogens, low production costs, fast turnaround and response times and rapid, nearly-unlimited scalability. Also, because dried leaves and seeds can be stored at room temperature for lengthy periods without loss of recombinant proteins, plant expression systems have the potential to offer lucrative benefits from the development of edible vaccines and prophylactics, as these would not require “cold chain” storage and transportation, and could be administered in mass volumes with minimal processing. Several biotechnology companies currently have developed and adopted plant-based platforms for commercial production of recombinant protein therapeutics. In this manuscript, we outline the challenges in the process of livestock immunization as well as the current plant biotechnology developments aimed to address these challenges.

Purification of plant-derived antibodies through direct immobilization of affinity ligands on cellulose.

Plants possess enormous potential as factories for the large scale production of therapeutic reagents such as recombinant proteins and antibodies. A major factor limiting commercial advances of plant-derived pharmaceuticals is the cost and inefficiency of purification. As a model system, we have developed a simple yet robust method for immobilizing affinity capture ligands onto solid supports by interfacing the secreted expression and coupling of a chimeric fusion protein in Pichia pastoris to microcrystalline cellulose in a single step. The fusion protein, which consisted of antibody-binding proteins L and G fused to a cellulose-binding domain (LG-CBD), was tethered directly onto cellulose resins added to P. pastoris cultures and subsequently used for antibody purification. Both the antibody-binding protein L and protein G domains were functional, as demonstrated by the ability of cellulose-immobilized LG-CBD to purify both a scFv antibody fragment from yeast and a human IgG1 monoclonal antibody from transgenic tobacco. Furthermore, combining two P. pastoris strains expressing LG-CBD and scFv with CP-102 cellulose in a single culture allowed for easy recovery of biologically active scFv. Direct immobilization of affinity purification ligands, such as LG-CBD, onto inexpensive support matrices such as cellulose is an effective method for the generation of functional, single-use antibody purification reagents. Straightforward preparation of purification reagents will help make antibody purification from genetically modified crop plants feasible and address one of the major bottlenecks facing commercialization of plant-derived pharmaceuticals.

Identification of the full-length Hs1pro-1 coding sequence and preliminary evaluation of soybean cyst nematode resistance in soybean transformed with Hs1pro-1 cDNA

The Hs1pro-1 gene reportedly confers resistance to the beet cyst nematode in wild beet and sugar beet. Here, we tested the hypothesis that Hs1pro-1 confers resistance in soybean against the soybean cyst nematode (SCN). The full-length Hs1pro-1 coding sequence, which encodes a predicted polypeptide of 490 amino acids, was first acquired then expressed in ‘Westag’ soybean using a constitutive octopine synthase – mannopine synthase promoter. Thirty T0 lines that successfully expressed the Hs1pro-1 gene, as indicated by both polymerase chain reaction and reverse transcriptase – polymerase chain reaction analyses, were generated. Bioassay of the T1 progeny from these lines revealed that only five T0 lines grew normally and exhibited a high degree of SCN resistance. On average, these T1 transgenic progeny were about 70% more resistant to SCN than susceptible control cultivars. These preliminary data suggest that Hs1pro-1 is a promising candidate for genetically engineering SCN resistance in elite, locally adapt...