6: Recombinant DNA expression systems Flashcards
Identify 3 enzymes that manipulate DNA and explain what they do.
Restriction endonucleases - Cleave DNA at specific base sequences
DNA ligase - Joining 2 DNA molecules/fragments
DNA polymerase - Adds nucleotides to the 3’ end using the second strand as a template.
Name some protein based pharmaceuticals that are available.
Anticoagulants - Eg Hirudin, Heparin
Thrombolytic agents - Eg Urokinase, Streptokinase
Diabetes treatments - Eg Insulin
Erythropoiesis (red blood cell production) - Eg Erythropoeitin
Chemotherapy - Eg Asparaginase
What is a restriction endonuclease and what does it do?
A restriction endonuclease is an enzyme that recognises and cleaves specific DNA sequences known as restriction sites (4-8base pairs) to generate a reproducible set of fragments called restriction fragments.
Endonucleases cleave within the DNA, while exonucleases cleave at the end of DNA.
The recognition of the sequence to be cleaved is mediated by the 12 hydrogen bonds between purines (A+G) and the 6 amino acid residues (Glu + 2xArg per subunit) in the dimeric enzyme.
These enzymes are isolated from bacteria and are used to fight against viral infections.
How does DNA ligase work?
DNA ligase joins strands of DNA by catalysing the formation of covalent phosphodiester bonds between the phosphate at the 5’ end of one nucleotide to the hydroxyl at the 3’ end of another nucleotide.
What is the use of DNA cloning?
DNA cloning involved the isolating and amplification of a specific DNA sequence. The amplification of these sequences can essentially produce an unlimited amount of that DNA, which can result in an over-expression of a protein (this can be desired or undesired)
This process is useful for protein purification, structural & functional studies, inactivation of a gene and in protein based pharmaceuticals.
What is a plasmid and what are some plasmid vectors?
Plasmids are independently replicating DNA located in some bacterial cells. Their structural feature include; ORI, Ampr and regions for DNA insertion.
Plasmid vectors:
- Circular for replication
- Contain ‘ORI’ (Origin of replication) that DNA polymerase binds to, to initiate replication
- Carry a selectable marker Eg. drug resistance (Ampr)
- Multiple cloning sites i.e. Regions for DNA insertion.
- Colour selection to detect recombinant inserts. Eg Blue/White Beta-Galactosidase system
What are the different cloning vector types that differ depending on the size of DNA?
- Plasmids: Up to 5Kb (Kb = Kilobases = 1000 bases)
- Phage Lambda: Up to 50Kb
- Bacterial artificial chromosome (BAC): Up to 300Kb
- Yeast artificial chromosome (YAC): Up to 2000Kb.
Expression vectors make RNA and protein from inserted DNA. Shuttle vectors are able to grow in bacterial and mammals.
Briefly describe the cloning process.
The plasmid is cut with restriction endonucleases, producing ‘stick ends’. The foreign DNA is cut by the same restriction endonuclease, hence producing matching ‘sticky ends’. When the plasmid and the foreign DNA is mixed, these ends attach and are joined by DNA ligase. This creates a recombinant molecule that can be transferred into bacterial cells using heat and calcium chloride. This enables the transfer of antibiotic resistance.
Explain how the cloning process plays a role in in vitro mutagenesis.
When the role of a specific amino acid within a protein is determined, these functional effects within a cell culture can be observed allowing for random substitutions/conversions to occur.
This conversion/substitution involved removing a site so that a mimicking site can be added. Eg Erythropoietin (EPO)
What is the EPO gene and protein?
The EPO gene is located in the long arm of the 7th chromosome, primarily in adult kidneys or fetal livers.
The EPO protein consists of 27 amino acid signal peptides. The mature protein is 166 amino acids long, consisting of 3 potential N-linked glycosylation sites.
Describe the expression vectors for recombinant EPO
Recombinant EPO is expressed using the entire gene, due to its relatively short size (3-4kb)
The expression vectors include:
- AmpR (Ampicillin resistance gene): found in the plasmid of E.coli
- DHFR (Dihydrofolate reductase gene): found in the plasmid of mammalian cells.
- SV40 ORI (Simian virus 40 is a strong promoter)
