Pharmaceutics - Bolhuis Flashcards
When producing Insulin, Factor VIII and Antithrombin (ATryn), what hosts would you use?
Insulin –> E.coli/yeast
Factor VIII –> Mammalian Cells
Antithrombin (ATryn) –> Goats
In which direction do both genes and Polymerases run?
5’ –> 3’
What’s the difference between monocistronic and polycistronic
Monocistronic –> One genes forms 1 mRNA, forming 1 polypeptide
Polycistronic (Operon) –> 2 or more genes forms 1 mRNA, forming 2 or more polypeptides
How is mRNA proccessed in eukaryotic cells?
Introns are spliced out
5’ 7-methylguanylate cap (m7Gppp)
Polyadenylation adds polyA tail after the stop codon
What are the 3 natural proccess of DNA transfering?
Transformation –> Uptake of free DNA
Known as transfection in animals
Conjugation –> Transfer of DNA via cell-cell contact
Transduction –> Transfer of DNA mediated by a virus
What are the 3 main stages in molecular cloning?
Isolation of source DNA
Inserting source DNA into cloning vector
Introduction of cloned DNA into a host organism
CaCl2 and a heat pulse required
How does PCR work?
Denaturation of DNA stands –> 30s at 94 degrees C
Annealing primers –> 30s at 55-65 degrees C
Elongation with thermostable DNA polymerase (taq polymerase) –> At 72 degrees C
These are repeated for 25-35 times
After n number of cycles of PCR, how many DNA copies are made?
How do Restriction Enzymes work?
They cut DNA at palindromes sequences (restriction sites)
This creates both sticky/blunt ends
How does DNA Ligase work?
An ATP dependent enzyme that sticks sticky ends together
What are the 3 important regions in a plasmid for cloning?
Replication origin
Selection marker –> eg, genes for antibiotic resistance or for growth on certain media
Region where DNA can be inserted
What is ‘Blue-White Screening’?
A way of finding out if the foreign DNA has been inserted into the MCS (multiple cloning site)
The MCS is in the lacZ gene (which encodes for a B-galactosidase)….so if this is inactive, the foreign DNA is present
Blue Colour = Colonies with intact lacZ –> as B galactosidase converts X-gal into blue colour
White Colour –> Foreign DNA in the MCS (lacZ gene), which inactivates B-galactosidase
What are the 4 types of ‘other’ vectors?
Shuttle –> Plasmids that can replicate in at least 2 different hosts
Intergration –> Can’t replicate, but intergrate into chromosomes
Useful for knockouts
λ Cloning –> Can accomodate larger inserts
Artificial Chromosomes –> Can contain very large inserts, and so used for cloning large genes
What are beneficial properties for hosts for cloning and expression?
Grows rapidly in inexpensive media
Non-pathogenic
Genetically stable
Easily takes up DNA
Allows replication of the vector
Allows high levels of gene expression
What are the 2 different ways to recombinantly make insulin?
Method 1 –> Clone insulin A/B chains seperatly in E.coli as fusions with gene encoding B-galactosidase
Purify and cleave off B-galactosidase
Combine the 2 chains and refold in oxidisng chains in-vivo –> to make disulphide chains
Method 2 –> Close the proinsulin gene, and fuse to B-galctosidase in E.coli
Extract, purify and remove the B-galactosidase
Refold the proinsulin
Cleave the proinsulin with enzymes
Why can’t we use E.coli to make Factor VIII and antithrombin?
As they are heaviliy glycosylated
How is Factor VIII produced?
Continuous cell culture in large vessels
Then purification
