Microbial biotechnology 2

• Why is E.coli used as an expression system

  They have a short doubling time and so can be cultured in large amounts.

  They have high copy plasmids meaning that there are a large number of plasmids per cell.
• How can you optimise E.coli for protein expression

  By producing a mutant strain that is protease deficient.
• How can you optimise the cDNA for protein expression

  Codon usage optimisation improves the translation rate and stabilises the mRNA.
• What are the methods to optimise the purification of a protein in E.coli

  Fusion tags for affinity purification.

  Signal peptides promotes secretion.

  Chaperone co-expression assist protein folding.
• What does the replication origin do

  Controls the copy number
• Why do you need to optimise codon usage to improve the translation rate

  Different organisms favour different codons and so different tRNA molecules are found in different proportions.
• What are the methods to optimise codon usage

  Use mutant E.coli strains that overexpress the rare tRNAs.

  Replace the human-favoured codons with bacteria-favoured codons.
• How are human-favoured codons replaced with bacteria-favoured codons?

  cDNA is created and then denatured. Anneal the mutagenic primers and amplify using PCR. The mutagenic primers are favoured by bacteria.

  Remove the human DNA by methylating and then digesting with an endonuclease.

  Clone the mutated plasmid in E.coli. Amplify and purify.
• What does the incorporation of N-terminal fusions do

  It improves the translation rate by correcting code bias and increasing expression.

  Increases protein stability for small proteins. The addition of amino acids increases the protein half-life.

  Facilitates purification by promoting secretion [using signal peptides] allows for affinity purification.
• How are N-terminal fusions removed

  The N-terminal fusions are attached to the protein using a target sequence for proteases.

  Thrombin cleaves the protease target sequence after purification.
• What is a fusion tag 


  They are additions to the C or N terminal. They facilitate purification of recombinant proteins.
• How is a polyhistidine tag used as a fusion tag

  Used for affinity purification of an immobilised nickel chromatography column.

  Polyhistidine tag binds to the nickel column.

  Cleave and elute using imidazole.
• What is codon bias

  The degeneracy of the genetic code means that different organisms favour different codons.