Lecture 21 - Biotechnology in use Flashcards
Recombinant DNA technology pathways
Therapeutic products
Genetically modified products
Diagnosis
Energy applications
Financial reasons and biochemical reasons for choice of host for protein production
Financial - bacteria best and transgenics worst
Biochemical - mammalian best and bacteria worst
Insulin, post translational modifications and what it is best produced in?
PTM = disulphide bonds
Best produced in? - A and B chains separately produced in bacteria (this is the most economical way to produce it) - this is used for diabetes treatment
EPO, post translational modifications and what it is best produced in?
PTM = Glycosylation (addition of carbohydrate residues)
BEst produced in? - This cannot not be in bacteria if you want a functional EPO so it is best produced in mammalian cells because they have the enzymes available to glycosylate proteins (it doesn’t mean you can’t produce it in the other but in a mammalian cell culture it is the most cost effective way of doing it)
Antithrombin (AT), post translational modifications and what it is best produced in?
PTM = Gamma-carboxylation
Best produced in? - Transgenic animals are the best way even though they are expensive
Eukaryotic expression system using whole animal/transgenic animals
Advantages - can perform all types of post translational modifications, easy extraction of therapeutic protein from animals’ milk is possible, large quantities of therapeutic protein are produced, larger animals are more similar to ourselves and they will ensure that larger and more complex proteins fold correctly
Disadvantages - expensive, time and labour intensive, some approaches require sacrifice of the animal (e.g. the spleen is the only place that antibodies can be taken for example)
Transgenic animals where the gene’s expression is regulated by a milk-specific promoter allows easy isolation of proteins ( and in large quantities)
Recombinant Antithrombin
Antithrombin (AT) is an anti-blood clotting factor and activates blood clotting when appropriate and preventing it when it is not needed. AT deficiency leads to too much/inappropriate blood clotting
Eukaryotic expression vector ….
Tissue specific eukaryotic host cell promoter (e.g. goat mammary gland specific) to which host cell RNA polymerase binds to initiate transcription of human gene, but only in mammary glands of the animal, to drive target gene expression
Target genes (e.g. human AT gene)
Ori
Antibiotic resistance gene (selective marker)
Providing a permanent treatment
Recombinant DNA technologies in the clinic = recombinant proteins that require repeated administration of drugs for the life of the patient/ illness
How can we provide a permanent treatment? Use patients as the host for protein production! - this would be a one time treatment that would have positive lifestyle and health effects on the patient
Gene therapy
Plasmids can be carried into patients using viral vectors = gene therapy
Promoter allows for specific expression at a particular place e.g. might be liver specific. Provide a functional copy of the gene as well
Range of different viral vector types and each one has a different affinity/tropism for different cell types. Plasmid will effectively be integrated into the genome of the patient and you should now get expression for life of the new functional copy of the gene and therefore correct the disease.
Use a virus to introduce a plasmid effectively into the cells. The viral vector has deleted all of its genetic material which allows the plasmid to be carried efficiently into the cell of the body
Conventional biotechnology vs gene therapy
Conventional biotechnology - requires you to go back in day after day and month after month depending on the stability of the protein (not a permanent one time fix)
Gene therapy - introduces the plasmid/gene that is used to make the recombinant protein and this is generally a one off treatment because the virus information allows for stable integration into the host cell and generally into the chromosomes of cells that are affected
Could gene therapy permanently treat Type 1 diabetes?
Problem:
Beta cells in pancreas destroyed = autoantibodies
recognise proteins on beta cell surface
= no insulin produced.
Solution? - Turn liver cells into insulin making machines.
Insert a plasmid to do this ….Include a glucose responsive promoter (need promoter to drive the expression in the liver, need all the appropriate transcription facts that will allow glucose responsive expression of insulin within the liver), Pre-pro insulin cDNA (this is the gene, transgene), also an antibiotic resistance gene - this plasmid is inserted into a viral vector which will have a liver tropism and will act to make liver cells into insulin making machines
Prokaryotic expression system using bacterial cells/bacterial cell culture
Advantages - quick, cheap, free of eukaryotic pathogens
Disadvantages - incorrect folding of complex proteins, bacterial cells cannot perform post translational modifications on protein
A problem when making/expressing eukaryotic proteins using prokaryotic host cells is that the eukaryotic gene sequence contains intron sequences but the bacterial host cell doesn’t have an “intron removal machinery” (as bacterial genes do not have introns). So, before the DNA of the gene of interest can be placed into the cloning vector, a DNA version of the gene that do not contain the intron sequences must be generated (through a process called reverse transcription (of the intron-less spliced mature mRNA). Reverse transcription converts single stranded mRNA molecule into a double stranded DNA molecule.
Eukaryotic expression system using eukaryotic animal cells/eukaryotic animal cell culture
Bacterial cell carrying cloning plasmid with you gene of interest. Eukaryotic promoter - no gene expression in bacterial cell. Ori ensures that a large number of plasmids are made. Extract plasmids from transformed cells and analyse the sequence fo the gene of interest on some plasmids to confirm that it carries the desired DNA sequence. Analysis by methods such as PCR, DNA gel electrophoresis, restriction enzyme digest and sequencing. Move DNA (plasmid with eukaryotic promoter) into eukaryotic cell using non-viral methods, this is transfection
Advantages - can perform some types of post translational modifications
Disadvantages - cannot perform some types of post translational modifications
