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.
The biophysical and chemical properties of TrypZean and native bovine trypsin are compared in Table I. TrypZean has the same
amino-acid sequence as the bovine-sourced product. Functionally, its activity appears to be identical to that of the native
bovine protein. However, pancreatic trypsin is not glycosylated, whereas characterization of the maize-derived trypsin has
shown that corn glycosylates the enzyme. It was important to pinpoint precisely where the protein was glycosylated, so that
the exact structure could be known for pharmaceutical applications, and to fully understand the biological processes. To provide
this full characterization, a novel way of preparing the samples was developed.
Table I: Physicochemical properties of native bovine trypsin and TrypZean.
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
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).