Differences between prokaryotic and eukaryotic drug production systems Flashcards
Limitations of prokaryotic expression systems
Some eukaryote proteins are unstable when expressed in bacteria or lack biological activity
Prokaryotes lack the ability to undertake the following post-translational modifications: glycosylation, proteolytic cleavage, disulphide bonds
Recombinant proteins expressed at high levels are stored as inclusion bodies so need to be refolded
Prokaryotes harbour pyrogens such as LPS and thus the final product requires extensive purification
Bacterial protein production and secretion
Proteins are mainly retained within the body of the bacteria
Secreted proteins are rare
Why is post translational modification important in drug design?
It affects the shape of the final protein
Influences biological activity e.g. antibodies, enzymes
Affects half life- misfolded proteins are degraded by proteases, protein with the wrong sugar groups such as antibodies are removed from the circulation
Glycosylation
~50% of all eukaryotic proteins synthesised in the rough ER are glycosylated, enzyme directed site specific process as opposed to the non-enzymatic chemical reaction of glycation
N linked glycosylation is to the amide nitrogen of asparagines side chains
O linked glycosylation is to the hydroxy oxygen of serine and threonine side chains
Glycosylated protein products
Currently > 100 protein products approved in USA and Europe
Products include full length IgG antibodies, hormones, EPO and G/M-CSF etc.
Failure to glycosylate a protein with the correct combination of sugars will alter its molecular size and charge, immunogenicity and pharmacological activity
Post translational modification of insulin
Cleavage of signal peptide by signal peptidase
Disulfide bond formation
Cleavage by trypsin-like enzymes releases the C peptide
The C chain is packaged in the secretory vesicle and is secreted along with active insulin
Further trimming by a carboxypeptidase B-like enzyme removes two basic residues from each of the new ends
Disulphide bond formation
Disulphide bonds in proteins are formed between the thiol groups of cysteine residues
It stabilizes protein structure by linking protein strands to help form the final structure and by forming hydrophobic regions
In eukaryotic cells, disulphide bonds are generally formed in the lumen of the RER
Mostly found in secretory proteins, lysosomal proteins and the exoplasmic domains of membrane proteins
Potential eukaryotic protein expression systems
Yeast
Insect cells
Mammalian cells
Transgenic animals/ plants
Yeast as a eukaryote system
Post translational processes similar to mammalian, can perform N-glycosylations
Tend to add sugars side chains of high mannose content- affinity for mamnose receptors
Genetics well known
Rapid growth
Listed as generally recognised as safe
Do not produce pyrogens such as LPS
Cell culture based systems
Chine hamster ovary cells are the mammalian cell of choice for production of glycoproteins
Advantages: produce N-glycans similar to those found in humans
Disadvantages: high cost, long development time from gene cloning to cell line, pathways similar but not the same as humans, propagation of infectious agents
Somatic cell nuclear transfer
Developed differentiated cells taken from organism to be cloned
Cell fused to unfertilised egg cell with nucleus removed
Begins to develop into a embryo
Implanted into host
