Cell Factories - Bacterial Systems

Question 1

Requirements for bacterial systems?

Answer 1

• 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

Question 2

Advantages of bacterial systems?

Answer 2

• Well characterised
• Genetically easy to manipulate
• Easy to culture in large volumes

Question 3

Disadvantages of bacterial systems?

Answer 3

• Unable to conduct PT processing that eukaryotic enzymes require for the activity
• Overproduction of recombinant proteins = misfolding and aggregation

Question 4

Promoters used in bacterial systems?

Answer 4

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.

Question 5

Problems in bacterial systems promoters?

Answer 5

In large vol culture = temp ^ is gradual & expensive

- ^ temp = misfolding of transgenic product & induces heat shock response = induction of endogenous proteases

Question 6

Metabolically regulated promoters in bacterial systems?

Answer 6

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

Question 7

Problems in metabolically regulated promoters in bacterial systems?

Answer 7

Works well on lab scale.

BUT IPTG = too expensive to be used in large-volume (industrial-scale) fermentors.

Question 8

PhoA gene

Answer 8

Encodes alkaline phosphatase. Promoter induced by phosphate starvation – can result in 103-fold induction of transcription

Question 9

Use of different polymerases in bacterial systems?

Answer 9

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.

Question 10

DSP in bacterial systems?

Answer 10

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

Question 11

DSP in bacterial bystems - E.coli

Answer 11

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

Question 12

How to obtain secretion in DSP in bacterial systems?

Answer 12

• 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)

Question 13

Affinity purification ligands - bacterial systems

Answer 13

• 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

Question 14

Transgene stability in bacterial systems

Answer 14

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

Question 15

Alternative methods for transgene stability in bacterial systems

Answer 15

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)

Question 16

Human insulin as a protein manufactured in E.coli

Answer 16

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.

Question 17

Additional E.coli dervived products by bacterial systems

Answer 17

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