R.O Lecture 4 Flashcards
Recombinant Protein Expression
Producing large quantities of recombinant proteins in host cells
Examples of biopharmaceuticals produced in bacteria
*Insulin
- Filgrastim (cytokine: stimulates hematopoiesis)
- Interferons (cytokines: treats lymphoma, leukaemia, kidney cancer etc.)
- Pegvisomant (human growth hormone analogue:
treats acromegaly)Insulin
Cloning Vectors vs Expression Vectors
- Cloning vectors are typically used to multiply the DNA insert in the host cell
- Expression vectors are used for expression of a gene of interest (produce protein)
Origin of Replication
- Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes.
- Without replication origin, DNA cannot be replicated in the cell.
- Common to both cloning and expression vectors.
Selectable marker
*Selectable marker is required for maintenance of plasmid in
the cell.
* Under the selective conditions, only cells that contain plasmids with selectable marker can survive
*Common to both cloning and expression vectors.
What type of genes are used as selectable markers?
Genes that make cells resistant to various antibiotics, like
ampicillin (ampr), tetracycline (tetr) or chloramphenicol (cat)
are used
Name the features of expression vectors
*Promotor - 2 sites: 10 (TATAAT) and 35 (TTGACA) upstream from ATG- Inducible promotor ( lac IPTG)
*Shine Dalgarno- Ribosomal binding site (RBS) AGGAGG between 4 and 14 upstream from ATG
*N and C Terminal Tag
*Fusion protein
*Protease cleavage site TEV; PreScission protease
*MCS
*Repressor controls ‘leaky’ promotor
What is Interleukin 2 (IL-2)?
*IL 2 is a cytokine; regulates activities of cells of the immune
system
*Recombinant IL 2 (Aldesleukin; Brand name Proleukin®)
*Protein therapeutic used for treatment of cancers (renal cell
cancer, metastatic melanoma)
Describe the process of making recombinant IL-2
Perform PCR to isolate and amplify the IL-2 gene
*Isolate template DNA
*Design primers with restriction sites to target PCR product to specific location in the plasmid MCS
*Design PCR program
N.B look at slides 8-13 for this process
- Isolate template DNA
The template has to be cDNA (not genomic DNA) as E.coli cannot handle intron-containing genes
cDNA is synthesised as complementary DNA to mRNA (using reverse transcriptase)
(Where do we get the mRNA) Isolate from cells that express IL-2, in this case should be from human peripheral blood lymphocytes (T Cells)
- Design Primers with restriction Sites
Design primers with restriction sites to target PCR product to specific location in the plasmid MCS
What does using the same restriction sites in the primers produce?
Produces complementary sticky ends: targets the PCR product to a specific location in the plasmid MCS
What are restriction enzymes?
Restriction enzymes cut the plasmid to allow insertion of the PCR product
How do we select restriction sites
Select restriction sites that occur within the MCS of your vector and don’t occur within the gene
Design PCR primers for human IL-2
Get mRNA/cDNA sequence (no introns!) from NCBI database.
PCR primers should be placed at the start and end of the (protein) coding sequence (cds).
Framed by START (ATG) and STOP (TGA) codons
