Post-translational modifications recombinant proteins

Yeast is the third expression system used to produce biopharmaceuticals. As mammalian systems, they possess the ability to cany out post-translational modifications of proteins, although the glycosylation pattern usually varies somewhat from the patterns observed on the native protein or on the protein expressed in mammalian cells. Two recombinant proteins expressed in Saccharomyces cerevisiae are now approved for general medical use hepatitis B surface antigen vaccine and the anticoagulant Hirudin . Alternative promising production systems, in particular transgenic animal and plant systems, are still in development but these systems have to prove that they are technically and economically attractive. 

When recombinant proteins are produced in a heterologous system, there may potentially be differences between the final product and the natural molecule. Hence, for each new protein produced in alfalfa, a thorough analysis of the processing, folding, assembly and post-translational modification is conducted to ensure the conformity of the purified molecules. This section describes the analysis of alfalfa-derived... 

One of the most important considerations for the improvement of protein yields is subcellular protein targeting, because the compartment in which a recombinant protein accumulates strongly influences the interrelated processes of folding, assembly and post-translational modification. All of these contribute to protein stability and hence help to determine the final yield [88]. 

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. 

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. 

Nucleolin lacks a characteristic DNA binding domain (Ginisty et al, 1999). Its non-specific affinity for DNA is conferred by two different domains its four RNA binding domains, particularly the 3rd and the 4th ones, and its C-terminal GAR domain (Hanakahi et al, 1999 Sapp et al, 1989). Of importance, these properties were determined in vitro with the native protein purified from cell extracts or recombinant truncated proteins they are likely to be altered in vivo by interaction with other DNA binding factors (Dempsey et al, 1998) and/or by post-translational modifications. 

As described above, histones are much more than passive structural players within chromatin. Dynamic post-translational modifications of these proteins confer specialized chemical proprieties to chromatin of both informational and structural nature with important functional implications. The highly conserved sites for acetylation, methylation, phosphorylation, ADP-ribosylation, and ubiquitination events on histone tails appear to orchestrate functional activities that range from transcriptional activation and repression to DNA repair and recombination. 

Intact plants are also suitable for cost-effective production of recombinant pharmaceutical proteins providing products free of contaminations with endotoxins or human pathogens. Plants generally promote the proper fold of foreign proteins and post-translational modifications that are somehow similar to those of mammalian systems. Also, production of vaccine candidates in cereal seeds allows antigen protection from proteolysis which ensures their stability for a long period of time. 

The protein products from recombinant plant cells may be more functional and potent as pharmaceuticals than those from microbial origin because a post-translational modification is likely to occur in plant cells. 

Mammalian cell culture is a technology used for the production of recombinant proteins of therapeutic use, as they can secrete proteins with post-translational modifications similar to those present in human proteins. The most important advantages of this capacity of the mammalian cell lines are that they secrete a protein with the similar characteristics to the original protein, so that the protein can be used for human treatment without generating immunological responses. 

---