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LT9 Recombinant Protein Production
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Recombinant Protein Production
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Recombinant Protein Production Most proteins in an eukaryotic cell are normally present in very small amounts –
DNA cloning and genetic engineering makes it possible to produce any protein in
very high amounts
Potential hosts…
- Bacteria
- Mammalian cells
- Yeasts
- Algae
- Crop plants
- Livestock
Types of recombinant proteins
1. Therapeutic proteins
Hormones, blood factors, insulin, erythropoietin, interferons, tissue plasminogen
activator, vaccines, antibodies
2. Diagnosis
Enzymes, antibodies, biosensors
3. Commercial Enzymes
Industrial – food/textile production
Biotech enzymes – restriction enzymes, thermostable polymerases, ligases
Purifying from natural source Recombinant protein production
Availability of starting material Can use non-pathogenic host that is
Abundance of protein within easily cultured
cell/tissue Genetic engineering allows high
Contamination/infection expression of required proteins
Ethical considerations Much reduced contamination and
Purification considerations infection risk
Cost Production may be more ethically
acceptable procedure
Production of Recombinant Proteins
High-level production usually accomplished by using specially designed vectors =
expression vectors
Expression vectors also include appropriate transcription and translation signals
so that an inserted gene is expressed at very high levels
, Expression vector replicated at each round of cell division, giving rise to a cell
culture able toproduce high amounts of protein of interst
Protein produced within the cell – must be purified by after cell lysis by
chromatography
As it is so abundant in the cell lysate (1-10% of total cell protein), purification is
usually easy to accomplish
Considerations
Gene Structure
Bacteria cannot remove eukaryotic introns
Start with cDNA of eukaryotic genes
Promoters: usually organism-specific
Use endogenous promoters – inducible
promoters/tissue-specific promoters
Codon Optimisation
- Genetic code is universal
- Code is degenerate (several codons code for the
same protein)
- Different organisms show preferences
- Need to re-engineer code to match preferences of
host organism
Synthetic Genes
PCR extension of seed oligonucleotides
Delivery Methods
Electroporation – electrical field is applied to cells
in order to increase the permeability of the cell
membrane
Chemical treatment
Biolistics – “gene gun”: millions of DNA coated metal
particles are shot at target cells or tissues
Microinjection
Expression Considerations
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