Biotechnology Flashcards
old insulin treatment
- Insulin purified from pancreas of cattle or pigs
- Human amino acid sequence similar but not identical
- Immune responses range from local irritation to anaphylactic shock
- Not always 100% pure
recombinant human protein formation process
- Get DNA encoding a protein of interest e.g. Insulin from pancreatic cells
- Clone DNA into an expression plasmid, along with promotor to bind transcription factors and RNA Pol to allow expression of DNA into RNA and AB resistance gene to protect bacteria against antibiotics
- Transfer plasmid into your favourite cell type (bacteria, yeast, mammalian, insect) or creature (plant or animal)
- Let the cells produce lots of protein
advantages of recombinant protein using bacteria
- Not contaminated with pathogens in pigs/cattle
- Makes pure human insulin
- Cheaper
- High yield
disadvantages of recombinant proteins using bacteria
- Proteins often partially folded
- Inability to perform post-translational modifications
production of insulin
- Insulin has no post-translation modifications therefore can be produced in bacteria, which cannot do PTM’s
- produced as two chains which are then connected by disulfide bridges afterwards
production of EPO in CHO cells
- erythropoeitin EPO, used as performance enhancing drug, requires glycosylation
- made in Chinese Hamster Ovary (CHO) cells in culture
- increase in RBCs leads to an increase in the oxygenation of muscle
3 methods of producing recomb human proteins
- produced in bacteria if no PTM’s needed
- produced in CHO cells if some PTM’s required
- produced in whole animals if all PTM’s required
why do we need recombinant EPO
- Many disease states result in lowered red blood cell counts
- chronic renal failure can cause a decrease in EPO levels, leading to anaemia (decrease in red blood cell levels)
- cancer treatments (chemotherapy) may lead to anaemia
advantages and disadvantages of whole animal recomb protein production
advantages:
- can do all PTMs
- easy extraction (via milk)
- large amounts of therapeutic protein produced
disadvantages:
- time, money and labour intensive
- requires sacrifice of animal
advantages and disadvantages of using eukaryotic animal cells (CHO cells)
advantages:
- can perform most types of PTMs
disadvantages:
- cannot perform some types of PTMs
antithrombin production using whole animals
- AT is anti-blood clotting factor and activates/regulates blood clotting when appropriate (by preventing when not needed)
- T protein expressed in the milk of transgenic goats at lactation
- AT then purified from other milk proteins
what is gene therapy
- taking plasmid with human gene and promoter and putting it directly into humans via a virus vector which protects it from body cells
- Permanent fix - inserting gene that encodes recombinant protein into body, rather than directing recombinant itself into body (temporary)
method of gene therapy
- Virus is coated in specific protein that allows us to target cell of interest
- Often HIV virus is used - must delete HIV genome and add plasmid and promoter
- Virus infects cell and introduces corrected gene (+ promoter) into cell chromosome
- Results in correct human protein being produced
treating type I diabetes using gene therapy
- Beta cells in pancreas destroyed = autoantibodies recognise proteins on beta cell surface = no insulin produced.
- Virus carrying corrected gene and promoter has surface proteins specific to liver cells, which target liver cells and turn them into insulin making machines
what is Cas9
- enzyme that cleaves DNA – double strand breaks
- dependant on guide RNA binding to complementary sequence
- normally double strand break is repaired though non-homologous end joining but if a donor sequence is present in nucleus then homology directed repair can occur which fixes mutations
gene editing (targeted gene therapy)
replaces whole abnormal gene with intact version thereby correcting mutation using Crispr-Cas9 tool
how does Crispr-Cas work
- guide RNA with matching genomic sequence bidns downstream of PAM
- cleavage by Cas9 protein
- insertion of Donor DNA by viral vector
treating muscular dystrophy using gene editing
- Take stem cells and add Crispr-Cas and guide RNA to target dystrophin
- Crispr-Cas cuts dystrophin and inserts gene, followed by non-homologous end-rejoining
non-homologous end-joining
- when donor sequence not present
- often results in mutations
what goes wrong in melanoma
UV light causes DNA to melanocytes, causing mutation which isnt repaired so melanocytes undergo uncontrolled cell division, resulting in melanoma
melanocytes
- found in epidermis of skin, eyes
- produce melanin containing melanosomes for delivery to keratinocytes
- melanin production activated by UV
melanin
- two types: pheomelanin = red, eumelanin = black or brown
- protect DNA against UV damage
- redheads have recessive mutation in MCR1 gene so no pheomelanin converted to eumelanin
tyrosinase and albinism
- enzyme that converts tyrosine to DOPA so pathway of production of melanin can take place
- Albinism = mutation in tyrosinase prevents enzyme function so no melanin produced
antibodies for treatment
- antibodies are increasingly being used as therapeutic agents
- exploit the specificity of antibodies
- most therapeutic antibodies target either:
- cancer e.g. Herceptin (for breast cancer)
- Or immune system e.g. Keytruda (for melanoma)
antibody structure
- has fixed Fc and variable Fab region
- Each domain (in both H and L chains) is made up of two antiparallel bsheets, linked by a disulphide bond
- The variable domains (in both H and L chains) contain complementarity Determining Regions - hypervariable regions
- Antigen-antibody interactions are mediated by the non-covalent interaction of amino acid side chains
T cell
T cell = a type of lymphocyte (white blood cell) involved in cell-mediated immunity
PD-L1
protein on tumour cell which acts as ligand and prevents PD1 on T-cell from detecting attacking tumour cell so T cell cannot destroy it
how to block PD1
antibody that binds to PD1 (i.e. anti-PD1) will prevent PD-L1 from binding. Blocking PD1 will activate T cell and kill tumour
process of making anti-PD1 antibodies
- Inject mouse with PD1
- Mouse will produce immune response
- Antibodies released against PD1
- Isolate antibodies and sequence them, look at AA sequence and work out sequence of CDR
Beer’s law
absorbance increases as solute increases
lambert’s law
Absorbance increases with increasing path length
beer-lambert law
A= ε.c.l
