Recombinant bovine trypsin has been produced in transgenic maize as a pathogen-free alternative to the animal-derived reagent. Biological reagents intended for pharmaceutical purposes require comprehensive analysis including detailed information about sequence and posttranslational modifications. In this study, several techniques, including mass spectroscopy (MS) analysis of the intact protein, peptide mapping, and MS analysis of released glycans were applied and an in-depth characterization of maize derived trypsin was achieved, revealing an unusual nonconsensus N-linked glycosylation.
The protease enzyme trypsin is produced in the pancreas and has many uses in the pharmaceutical sector. As well as being used to digest proteins for laboratory analysis, where it specifically cleaves at arginine and lysine residues, it also has applications in pharmaceutical production, such as producing insulin from pro-insulin and making vaccines. For any pharmaceutical process, it is essential that the enzyme is free from pathogens, which may be present in animal-derived products.The increasing desire of industry to avoid reagents from animal sources has inspired many attempts to express bovine trypsin in alternative platforms. For that reason, the expression of trypsin in maize for large-scale industrial and pharmaceutical applications was developed and optimized by Woodard et al. (1). The task was accomplished by expressing the enzyme in an inactive zymogen form that accumulates in the endosperm of the maize seeds. The zymogen gene was inserted into maize plants and cultivated in open fields. The purified enzyme is currently commercialized by Sigma-Aldrich under the trade name TrypZean. While more expensive than animal-derived trypsin, its cost is more than offset by the elimination of regulatory costs associated with viral-clearance studies that are needed when using the animal product.
GLYCOSYLATED PROTEIN ANALYSIS STRATEGIES
Glycosylation is a common post-translational modification, with around half of all human proteins bearing some form of sugar functionality. There are two major forms of glycosylation, O- and N-linked. For O-glycosylation, the sugar can be attached to the hydroxyl group of a serine or threonine residue anywhere in the protein. For N-glycosylation, a well-defined rule states where in a protein sequence N-glycosylation can occur: it is always attached to the amide group of an asparagine residue that is followed first by any amino acid other than proline, and then either serine or threonine. This so-called consensus sequence does not occur in trypsin, which implied that TrypZean was O-glycosylated.
The following are the three standard methods for the analysis of glycosylated proteins using mass spectrometry (MS):
1. Liberation of the glycans from the protein by chemical or enzymatic means, followed by derivatization of the glycan before MS analysis
2. MS analysis of the intact protein with no pretreatment (i.e., top-down strategy)
3. Peptide mapping, with proteolytic digestion of the glycosylated protein followed by MS analysis of the resulting digest (i.e., bottom-up strategy).