Q/A Session 4/5 Flashcards
What is a receptor?
Protein molecule that receives chemical signals from outside the cell
What is a ligand?
Molecule/drug that binds to a receptor
When alcohol binds to the GABAa receptor what enters the cell?
Cl- ions into the cell
What is the role of HIV protease?
Cleave long proteins into fragements
What kind of inhibitor will a pharma drug be?
A competitive inhibitor
What do transcriptional factors do?
Determines when and in what cell a gene is transcribed to produce mRNA
What are transcription factors?
Proteins that bind to a specific DNA sequence and control rate of transcription
What is a promoter?
DNA sequence at which transcription factors bind and recruit RNA polymerase
What does a promoter determine?
If and how much a gene is transcribed
What 4 things does PCR require?
- DNA from cell
- DNA from nucleotides
- Primers with sequence that can base pair to region of amplification
- Taq DNA polymerase
What are the 3 steps of PCR
- Heat DNA to 95 degrees to separate DNA strands. Heat breaks the H-bond
- Cool to ~60 degrees to base pair a DNA primer. Primer=short DNA sequence of nucleotides complementary to strand
- Heat DNA to 72 degrees to allow Taq DNA polymerase to copy DNA.
What do tumour suppressor genes do?
Encode proteins that prevent uncontrolled cell growth
What do onco-genes do?
Promote cell division
What is a proto-oncogene?
A normal gene that when mutated promotes uncontrolled cell growth
What are the components of recomb DNA plasmids?
- Origin of Replication
- Allows initiation of replication using host DNA polymerase - Antibiotic Resistance gene
- Allows selection of cells containing plasmid - Promoter
- Drives expression of gene in cells with appropriate transcription factor machinery
What are the characteristics of plasmids?
- Circular pieces of dsDNA
- Replicate independently of host chromosomal DNA, but use host machinery
- Provide a benefit to host
What are restriction enzymes?
- Cut dsDNA into specific sequences creating sticky ends
2. Found naturally in bacteria-degrade foreign DNA
What is transformation?
Transfer of plasmids into bacteria
What are the 5 steps of producing recombinant protein in prokaryotes?
- Isolate gene of interest
- Clone into expression plasmid
- Transform bacteria for expression
- Grow cells expressing gene of interest
- Isolate and purify protein
Advantages of prokaryote systems
- Low cost
- high yield
- pathogen free
Disadvantages of prokaryote systems
- Proteins often partially folded
- Inability to perform post-translational modifications
Advantages of mammalian cell recombs
- Protein can be produced as pre-pro-protein and processed efficiently
- Secreted from cells: easier purification
Disadvantages of mammillian systems
More expensive
What are the 5 steps of producing recombinant protein in eukaryotes?
- Isolate cDNA
- Clone into eukaryotic expression plasmid
- Transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells
- Extract recomb from cell media.
- Purify
What is glycosylation?
Requires mammalian cells. Adding carbohydrates to residues
What is pharming?
Using whole animals to make recomb proteins
What is insulin best produced in?
Bacteria. A+B chains are produced separately.
The PTM is formation of disulfide bonds
What is EPO best produced in?
Mammalian cell culture. Hamster Ovaries
PTM is glycosylation
What is Antithrombin best produced in?
Transgenic animals
PTM is carboxylation
What does gene therapy use?
Viral vector for stable integration into host cell
->gene encoding recomb protein
