Lecture 6: Recombinant DNA expression Systems Flashcards
What is needed to make protein-based pharmaceuticals?
Enzymes to manipulate DNA: restriction endonucleases: cleave DNA at specific base sequences, DNA ligase: joining 2 DNA molecules or fragments, DNA polymerase: adding nucleotides to the 3’ end using the second strand as a template.
Cloning vectors: allow the amplification and propagation of DNA molecules of interest.
Give examples of some protein-based pharmaceuticals.
Protein based pharmaceuticals include anti-coagulants (heparin), thrombocytes agents (streptokinase), diabetes treatments (insulin), erythropoietin, and chemotherapy agents (asparaginase).
Describe restriction endonucleases.
Recognise and cleave specific restriction sites within DNA (usually 4-8bp). Generates restriction fragments.
Large family of proteins isolated from bacteria. Used by enzymes to fight viral infection. The size of the recognition sequence determines how many times they will cut the DNA.
Some restriction endonucleases like EcoRI form sticky ends, in which there is overhang of some AAs at the cleavage site, while others form blunt ends in which there is no overhang of AAs.
Give an example of a restriction endonuclease.
Eg EcoRI: sequence recognition is mediated by 12 H bonds formed between A and G bases I the recognition site of DNA, and 6 AA residues In the dimeric enzyme.
Describe ligase.
Ligase catalyses the formation of a covalent phosphodiester bond between the 5’ phosphate of one nucleotide, and the 3’OH of a nucleotide upstream.
Describe DNA cloning and explain why it is done.
Refers to the isolation and amplification of a specific DNA sequence. It is done as an alternative to protein purification, which is a more laborious process, and leads to the isolation of only very small amounts of protein.
Once cloned DNA is isolated, protein can be produced in essentially unlimited amounts (over expression of the protein).
Cloned DNA is used for structural and functional studies, inactivation of gene and protein based pharmaceuticals.
Describe plasmid vectors.
Circular DNA with three major sites: origin of replication (ORI), DNA insertion region and selectable marker. May also have a colour selection region.
ORI is the binding site for DNA polymerase to initiate replication.
Selectable marker may be a gene for drug resistance, eg AMPr. Non-transformed bacteria are killed when exposed to antibiotic.
DNA insertion region is also known as the multiple cloning site, and is where the gene for the protein of interest is inserted.
Colour selection is used to detect the recombinant insert, eg blue/white beta galactosidase system (LacZ gene).
Describe cloning vector types.
cloning vectors for different sizes of DNA:
- plasmids: up to 5kb
- phage lambda vectors: up to 50kb
- bacterial artificial chromosome (BAC): 300kb
- yeast artificial chromosome (YAC): 2000kb
Expression vectors which make RNA and protein from the inserted DNA: eg shuttle vectors which can grow in two different species.
Describe the basic cloning process.
- Plasmid is cut with a restriction enzyme which generates sticky ends.
- Foreign DNA is cut with the same restriction enzyme, generating sticky ends that are complimentary to those in the plasmid.
- Plasmid and foreign DNA are mixed, and the sticky ends anneal. DNA ligase joins them to create the recombinant molecule.
- Transformation: E. coli cells are mixed with the recombinant plasmids in the presence of Calcium chloride.
- E. coli cultured on agar containing ampicillin. Only transformed cells survive due to ampicillin-resistance gene in recombinant plasmids.
Describe the transformation of bacteria.
Use high efficiency transformation strains to uptake recombinant plasmids.
Transformation takes place in the presence of calcium chloride (mechanism unknown), and other divalent cations, such as magnesium sulphate.
Electroporation drives the charged DNA into the bacterial cells (short, high energy electric pulse causes brief opening of the cell membrane).
Why clone DNA?
Cloning process allows in vitro mutagenesis.
To determine the role of specific amino acids in the function of the protein.
Look for functional effects in cell culture systems
Can do random substitutions
Can do conversion of post-translational modification sites (remove, add-EPO- or mimic site-Asp or Glu on a phosphorylation site)
In what cells may recombinant proteins be expressed?
Mammalian cell lines (Chinese hamster ovary, baby hamster kidney) Bacteria (E. coli) Yeast (S. Cerevisiae, P. pastoris) Insect cells (for Baculovirus) Plants
Describe the EPO gene and protein.
GENE: located of the long arm of chromosome 7. Expressed primarily in adult kidney and foetal liver. First isolated and expressed in 1985.
PROTEIN: 27aa signal peptide, typical of secreted proteins. Mature protein is 166aa long. The calculated molecular weight of the unglycosylated form is 18 399da. It has 3 potential N-linked glycosylation sites.
Describe the expression of recombinant EPO.
Expressed using the whole gene, which is relatively short (3-4k bases)
Expression vector contains the following 3 important components:
- ampicillin resistance gene for the selection of plasmid in E. coli.
- dihydrofolate reductase gene (DHFR) for selection of mammalian cells containing the recombinant plasmid. The mammalian cell culture medium lacks thymidine or precursor, which can be made by DHFR, and is necessary for DNA replication.
-SV40 ORI: simian virus 40 strong promoter.
Briefly describe the isolation of recombinant EPO.
Chinese hamster ovary cells are transfected with expression vector.
Select for stable transfectants (cells with plasmid integrated into the genome)
EPO is purified from culture medium: has MW of 34.4kda, and 19kda after deglycosylation (EPO has large amount of carbohydrate content)
Recombinant EPO is biologically active.
