Glycosylation post-translational modification

Capability of consistent post-translational modification (glycosylation, sulfation). 

The selection of an expression system for a cloned gene depends on many factors host/vector stability, expression level, ease of scale-up, post-translational modifications (glycosylation, secretion, amidation, folding, proteolysis, phosphorylation, etc.), product immunogenecity, and heterogenicity. 

Especially promising is the capability of ECD for determining the sites of protein post-translational modifications. Glycosylation and phosphorylation moieties are often labile under standard activation modes. The resulting MS/MS experiments yield little information on modification sites. ECD studies suggest that bonds remote to the modification sites are more labile, and the modification remains intact for MS/MS experiments. This is especially true for modifications such as phosphorylation, ° acetylation,deamidation, ... 

While electrospray is used for molecules of all molecular masses, it has had an especially marked impact on the measurement of accurate molecular mass for proteins. Traditionally, direct measurement of molecular mass on proteins has been difficult, with the obtained values accurate to only tens or even hundreds of Daltons. The advent of electrospray means that molecular masses of 20,000 Da and more can be measured with unprecedented accuracy (Figure 40.6). This level of accuracy means that it is also possible to identify post-translational modifications of proteins (e.g., glycosylation, acetylation, methylation, hydroxylation, etc.) and to detect mass changes associated with substitution or deletion of a single amino acid. 

Both ChEs undergo several post-translational modifications, including glycosylation and glycosylphosphatidy-linositolation (GPI), phosphorylation and carbamylation. 

Post-translation modification Changes that occur to proteins after peptide-bond formation has occurred, e.g. glycosylation and acylation. 

Post-translational modification of proteins plays a critical role in cellular function. For, example protein phosphorylation events control the majority of the signal transduction pathways in eukaryotic cells. Therefore, an important goal of proteomics is the identification of post-translational modifications. Proteins can undergo a wide range of post-translational modifications such as phosphorylation, glycosylation, sulphonation, palmitoylation and ADP-ribosylation. These modifications can play an essential role in the function of the protein and mass spectrometry has been used to characterize such modifications. 

Plant-based production systems are now being used commercially for the synthesis of foreign proteins [1-3]. Post-translational modification in plant cells is similar to that carried out by animal cells plant cells are also able to fold multimeric proteins correctly. The sites of glycosylation on plant-produced mammalian proteins are the same as on the native protein however, processing of N-linked glycans in the secretory pathway of plant cells results in a more diverse array of glycoforms than is produced in animal expression systems [4]. Glycoprotein activity is retained in plant-derived mammalian proteins. 

Differences in post-translational modification (PTM) detail. Human therapeutic proteins produced in several recombinant systems (e.g. yeast-, plant- and insect-based systems Chapter 5) can display altered PTM detail, particularly in the context of glycosylation (Chapter 2). Some sugar residues/motifs characteristic of these systems can be highly immunogenic in humans. 

Technical advances facilitating genetic manipulation of animal cells now allow routine production of therapeutic proteins in such systems. The major advantage of these systems is their ability to carry out post-translational modification of the protein product. As a result, many biopharmaceuticals that are naturally glycosylated are now produced in animal cell lines. CHO and BHK cells have become particularly popular in this regard. 

Post-translational modifications, in particular glycosylation patterns, can be incomplete and/or can differ very significantly from patterns associated with native human glycoproteins. 

Mammalian cell culture is more technically complex and more expensive than microbial cell fermentation. Therefore, it is usually only used in the manufacture of therapeutic proteins that show extensive and essential post-translational modifications. In practice, this usually refers to glycosylation, and the use of animal cell culture would be appropriate where the carbohydrate content and pattern are essential to the protein s biological activity, its stability or serum half-life. Therapeutic proteins falling into this category include EPO (Chapter 10), the gonadotrophins (Chapter 11), some cytokines (Chapters 8-10) and intact monoclonal antibodies (Chapter 13). 

Most interferons have now been produced in a variety of expression systems, including E. coli, fungi, yeast and some mammalian cell lines, such as CHO cell lines and monkey kidney cell lines. Most interferons currently in medical use are recombinant human (rh) products produced in E. coli. E. coli s inability to carry out post-translational modifications is irrelevant in most instances, as the majority of human IFN-as, as well as IFN- 3, are not normally glycosylated. Whereas IFN-y is glycosylated, the E. coli-derived unglycosylated form displays a biological activity similiar to the native human protein. 

Adrenergic receptors are also subject to multiple post-translational modifications. Like dopamine receptors, they are glycosylated, palmitoylated and phosphory-lated on various residues. There are multiple consensus... 

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