A case study for plant-made pharmaceuticals comparing different plant expression and production systems.

Over the last decade, plant-based production of pharmaceuticals has made remarkable progress as the expression of a diverse set of proteins has been demonstrated in a range of plant crops. Although the commercial exploitation is still pending, today various plant-based expression technologies have reached significant milestones through clinical testing in humans. Each of the protein manufacturing platforms in plants has specific benefits and drawbacks. We have engaged in comparing some of these production systems with respect to their performance: protein yield and quality. Using a specific tester protein (aprotinin), it was shown that functional aprotinin can be manufactured in plants in substantial amounts, as illustrated in this chapter.

Translocation of aprotinin, a therapeutic protease inhibitor, into the thylakoid lumen of genetically engineered tobacco chloroplasts.

Aprotinin, a bovine protease inhibitor of important therapeutic value, was expressed in tobacco plastid transformants. This disulphide bond-containing protein was targeted to the lumen of thylakoids using signal peptides derived from nuclear genes which encode lumenal proteins. Translocation was attempted via either the general secretion (Sec) or the twin-arginine translocation (Tat) pathway. In both cases, this strategy allowed the production of genuine aprotinin with its N-terminal arginine residue. The recombinant protease inhibitor was efficiently secreted within the lumen of thylakoids, accumulated in older leaves and was bound to trypsin, suggesting that the three disulphide bonds of aprotinin are correctly folded and paired in this chloroplast compartment. Mass spectrometric analysis indicated that translocation via the Sec pathway, unlike the Tat pathway, led predominantly to an oxidized protein. Translocation via the Tat pathway was linked to a slightly decreased growth rate, a pale-green leaf phenotype and supplementary expression products associated with the thylakoids.

Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.

Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that were found to be noncompeting. Unlike vectors derived from the same virus, noncompeting vectors effectively coexpress the heavy and light chains in the same cell throughout the plant body, resulting in yields of up to 0.5 g of assembled mAbs per kg of fresh-leaf biomass. This technology allows production of gram quantities of mAbs for research purposes in just several days, and the same protocol can be used on an industrial scale in situations requiring rapid response, such as pandemic or terrorism events.

Glucosinolate and amino acid biosynthesis in Arabidopsis.

Enzymes that catalyze the condensation of acetyl coenzyme A and 2-oxo acids are likely to be important in two distinct metabolic pathways in Arabidopsis. These are the synthesis of isopropylmalate, an intermediate of Leu biosynthesis in primary metabolism, and the synthesis of methylthioalkylmalates, intermediates of Met elongation in the synthesis of aliphatic glucosinolates (GSLs), in secondary metabolism. Four Arabidopsis genes in the ecotype Columbia potentially encode proteins that could catalyze these reactions. MAM1 and MAML are adjacent genes on chromosome 5 at the Gsl-elong locus, while MAML-3 and MAML-4 are at opposite ends of chr 1. The isopropylmalate synthase activity of each member of the MAM-like gene family was investigated by heterologous expression in an isopropylmalate synthase-null Escherichia coli mutant. Only the expression of MAML-3 restored the ability of the mutant to grow in the absence of Leu. A MAML knockout line (KO) lacked long-chain aliphatic GSLs, which were restored when the KO was transformed with a functional MAML gene. Variation in expression of MAML did not alter the total levels of Met-derived GSLs, but just the ratio of chain lengths. MAML overexpression in Columbia led to an increase in long-chain GSLs, and an increase in 3C GSLs. Moreover, plants overexpressing MAML contained at least two novel amino acids. One of these was positively identified via MS/MS as homo-Leu, while the other, with identical mass and fragmentation patterns, was likely to be homo-Ile. A MAML-4 KO did not exhibit any changes in GSL profile, but had perturbed soluble amino acid content.