Lecture 21: PRODUCTION OF THERAPEUTIC PROTEINS WITH A NEW FUNCTION Flashcards
What influences the host choice for protein production?
Speed and cost (financial reasons) and glycosylation and folding (biochemical reasons)
What is the worst to best host with relation to speed?
transgenic, plants, mammalian, insect, yeast, bacteria
What is the worst to best host with relation to cost?
transgenic, mammalian, plants, insect, yeast and bacteria
What is the worst to best host with relation to glycosylation?
bacteria, yeast, plants, insect, transgenic, mammalian
What is the worst to best host with relation to folding?
bacteria, yeast, plants, insect, transgenic, mammalian
What is the PTM for insulin?
Disulphide bonds
What is insulin best produced in?
A and B chains in separate bacteria
What is the PTM for EPO?
Glycosylation
What is EPO best produced in?
Mammalian cell culture
What is the PTM for antithrombin?
Gamma carboxylation
What is antithrombin best produced in?
Transgenic animals
How can we make new proteins?
Use a plasmid with promoter (cell type specific), transgender and antibiotic resistance for amplification in bacteria.
What can we do to create mutations in proteins?
Amplify a known enzyme using degenerate PCR (with ineffective enzymes)
What can be done with mutations with improved function?
They can be added to a plasmid using a promoter specific to bacterial or mammalian cels depending on whether or not post-translational modifications are required
What do recombinant DNA technologies in the clinic require?
Repeated administration of drugs for the life of the patients/illness
How can we provide a permanent treatment?
Use patients as the host for protein production
What happens in gene therapy?
Plasmids are carried into patients using viral vectors as if pure plasmid entered, the body would degrade it.
What happens with the gene in the viral vector?
It is inserted into the host cell chromosome
What do viruses have?
Different types of tropisms to make them more effective to certain cells
What does conventional biotechnology use?
A recombinant protein
What does gene therapy use?
A gene that encodes the recombinant protein
What is the problem with permanently treating type 1 diabetes?
Beta cells in the pancreas are destroyed so the autoantibodies recognise proteins on beta cell surface so no insulin is produced
What is the solution to permanently treating type 1 diabetes?
Turn liver cells into insulin making machines
What is the promoter for treating type 1 diabetes?
Glucose responsive (when glucose levels are high) and also liver cell specific
What is the gene for treating type 1 diabetes?
pre-pro insulin cDNA
What is the tropism for the virus for treating type 1 diabetes?
Liver cells
