Maize (Zea mays)‐derived bovine trypsin: characterization of the first large‐scale, commercial protein product from transgenic plants

Abstract

Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal-derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial-level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large-scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal-derived trypsin in the processing of pharmaceutical proteins.

In Plant Activation: An Inducible, Hyperexpression Platform for Recombinant Protein Production in Plants

“…Using INPACT to direct the expression of an apoplast-targeted zymogen form of the enzyme, we were able to achieve levels of up to 196 mg/kg (dry weight) in tobacco leaves, 3 d PEA. This yield is more than comparable to levels achieved in maize (Zea mays) seed (58 mg/kg dry weight; Woodard et al, 2003). Human Vitronectin is a large, multifunctional glycoprotein found in blood, some tissues, and the extracellular matrix.…”

Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen‐related proteins

“…Advocates of germ targeting have used the embryo-preferred globulin-1 promoter and, as a result, they have reported higher expression levels of recombinant b-glucuronidase (GUS), 6 avidin, 7 fungal laccase, 8 bovine trypsin, 9 and brazzein 10 than when using constitutive or endosperm-preferred promoters. An added advantage for targeting expression to germ is the smaller amount of biomass entering downstream processing 11 because germ is only 11% of the kernel mass.…”

Plant Molecular Farming: Much More than Medicines

“…The trypsinogen was fully converted into active trypsin upon extraction apparently by autocatalytic processing and/or endopeptidases in the seeds. The maize-derived trypsin was functionally identical and physically similar to native bovine trypsin (V max , K m , pH optimum, stability, and inhibition by aprotinin and benzamidine), although unlike the native protein, it was also glycosylated (76). Further characterization revealed an unusual nonconsensus N-glycosylation site at Asp77 (77).…”

“…In plants, these issues have been addressed by expressing inactive trypsinogen in maize seeds under the control of the embryo-preferred globulin-I promoter and the optimized barley α-amylase signal sequence, resulting in a yield of 3.3% TSP (76). The progeny of this maize line produced 58 mg of trypsin per kilogram of seed.…”