Disease Therapeutics Flashcards
What are ligated to create recombinant DNA?
Sticky ends created by restirction enzymes
Process of creating recomb DNA using vector
Cut vector plasmid using endonuclease…cut DNA using endonuclease…anneal together…DNA ligase seals
Bacteral recombinant strategy
Take plasmid from bacteria, add human DNA to plasmid, reinsert into bacterial cell
What allows purification
Affinity Tags
GST tags and how they work
Fused to a given protein
Glutathione S-transferase
Stick to glutathione…elute with glutathione in order to get protein out
Other way to purify proteins
Use protease to cleave off the affinity tag
Example - MBP with amylose
Therapeutics for Type 1 diabetes problems
Zoonotic viral transmissions, allergic response, can’t get pure enough
Insulin generated through
Proinsulin cleavage into inculin and C peptide
How can you make insulin (think disulfide bonds)
Make alpha and beta chain separately…refold and oxidize cysteines
What else can we do with recombinant DNA besides make more copies of existing DNA?
We can alter DNA to make amino acid changes
Things we can vary with insulin
Onset of action, time to peak concentration, duration of aciton
Are there allergic response to recombinant human insulins?
yes
Transgenic animal example
Put B-lactoglobulin promoter in front of AAT gene and it will be in the milk of an animal
Disdavantage of using cadaver growth hormone?
Risk of CJD
HPV vaccine generation
Add genes encoding for capsids of the virus to the recominant plasmid of DNA…add to yeast cells…purify and inject
