Cell Factories - Bacterial Systems Flashcards
Requirements for bacterial systems?
- Require ^ levels of recombinant protein production = will use ^ly active promoters that can be switched on as desired.
- Transformation vectors should be stably maintained in culture = need to ensure DSP of product is straight forward
Advantages of bacterial systems?
- Well characterised
- Genetically easy to manipulate
- Easy to culture in large volumes
Disadvantages of bacterial systems?
- Unable to conduct PT processing that eukaryotic enzymes require for the activity
- Overproduction of recombinant proteins = misfolding and aggregation
Promoters used in bacterial systems?
Bacteriophage –derived
λPL and λPR = strong promoters – regulated by cI repressor.
Regulation temp-modulated using the temp-sensitive allele cIts857 = represses transcription at low (30 C) temp, but not at high (40 C) temp
cI857 gene = provided on a defective prophage, or cloned on same plasmid as the transgene.
- latter strategy is better = defective repressor effectively inhibits transgene expression at 37 C = tighter regulation.
Transgene expression induced by ^ temp of culture to 40 C.
Problems in bacterial systems promoters?
In large vol culture = temp ^ is gradual & expensive
- ^ temp = misfolding of transgenic product & induces heat shock response = induction of endogenous proteases
Metabolically regulated promoters in bacterial systems?
Regulated by metabolites
Lac = weak promoter
Trp + Lac hybrid = ^ levels of protein production (Ptrp) regulated by addition of synthetic Lac inducer, IPTG = Tac promoter system
Problems in metabolically regulated promoters in bacterial systems?
Works well on lab scale.
BUT IPTG = too expensive to be used in large-volume (industrial-scale) fermentors.
PhoA gene
Encodes alkaline phosphatase. Promoter induced by phosphate starvation – can result in 103-fold induction of transcription
Use of different polymerases in bacterial systems?
Bacteriophage RNA polymerases = more active than the endogenous E. coli RNA polymerase.
Can express these polymerases as transgenes to elicit transcription of transcripts driven by phage promoters.
Bacteriophage T7 RNA polymerase = frequently used for this
- Can express the polymerase under the control of a tightly regulated promoter = well-regulated, high-level transgene expression.
DSP in bacterial systems?
Must be able to isolate transgenic product in soluble form easily
BUT if protein is secreted from cell b/ this makes intracellular proteases = easier to purify b/ less proteins in medium
DSP in bacterial bystems - E.coli
Does not secrete proteins well.
Protein secreted = remove N-terminal formyl-methionite residue (terminal is bacterium specific so not good for pharma product)
ALSO - once polypeptide reaches periplasm ^ chance of folding correctly b/ of chaperones in protein secretion
How to obtain secretion in DSP in bacterial systems?
- Encode transgene as a fusion product w/ a secretion signal that includes large proportion of native bacterial protein = better folding, improved solubility of transgene product, reduces proteolysis
- B/ translation product recognised as ‘self’ = allows removal of N-terminal formyl met residue w/ rest of protein.
If C-terminal fusion = allows purification strategy (e.f immunoaffinity chromatography using ligand that recognises native polypeptide)
Affinity purification ligands - bacterial systems
- antibodies ( e.g Myc, HA, FLAG tags)
- specific compounds recognised by native enzymes (Glutathione-S-transferase binds glutathione-sepharose)
- other chemical ligands (e.g. Nickel-agarose is used to purify polypeptides tagged with His6 tags)
More than one tag can be used to purify a polypeptide. - e.g an N-terminal His-Tag and a C-terminal FLAG-tag allows a first-round purification on a Ni-agarose column, immediately followed by a second round of affinity chromatography on an anti-FLAG immunoaffinity resin
Transgene stability in bacterial systems
VERY important
Plasmids can be lost from cultures = takeover” by non-transgene-containing cells.
So if plasmids are used as vectors, stringent selection required
B/ of the expense involved, antibiotics are NOT appropriate in industrial-volume cultures
Alternative methods for transgene stability in bacterial systems
par sequence in the plasmid
- par locus ensures segregation of E. coli chromosome into the two daughter cells at each cell division
OR
can use a mutant strain of bacterium in which a vital cellular function is provided on the vector.
-e.g SSB gene encoding the single-stranded binding protein (if an ssb- host is used, only cells containing the wt gene on a plasmid can grow)
Human insulin as a protein manufactured in E.coli
First transgenically produced pharmaceutical product.
Complex process b/ active insulin comprises two polypeptide chains associated by disulphide bridges.
Transgene provided as the preproinsulin cDNA sequence expressed as a C-terminal fusion with the E. coli tryptophan synthetase gene.
- Following growth in large-scale fermentation vessels (40m3), the protein is isolated as an insoluble inclusion body.
- This is purified by (i) cleavage from the native E. coli protein by treatment with cyanogen bromide (CNBr), which cleaves C-terminal to methionine residues.
- Disulphide bonds are cleaved by oxidative sulphitolysis, and the maintenance of a reducing environment to ensure that inappropriate S-S bonds do not form.
The formation of correct S-S bridges is then promoted by a period of oxidation at high pH, followed by a final proteolytic cleavage to generate the active two-chain mature protein.
Up to 2.5 milligrams of human insulin can be isolated from a 40m3 culture.
Additional E.coli dervived products by bacterial systems
Growth hormones
– Human GH used to treat growth defects
-Bovine & porcine GH used in agricultural production systems = Insulin-dependent growth factor – erythropoietin
used to treat anaemia – particularly following recovery from chemo for leukaemia: also abused by athletes
