338 #10

• Recombinant protein expression


  The process of producing a protein in large quantities by hijacking a host cell's machinery
• To study a protein requires some means of producing the protein in sufficient quantity and purity
• Many techniques used to study proteins require milligram quantities, which is actually quite a lot
• Extraction and purification from the natural source only yields very small quantities, and scale-up is impractical
• The solution is to hijack biology to produce large quantities (overexpression) of the protein
• Requirements for recombinant expression


  • Genetic material (DNA encoding the protein)
  • Ability to insert the DNA into the host cell
  • Ability to select for cells containing the DNA
  • Ability to control when the protein is expressed

  
• Vectors: plasmids


  DNA molecules used to introduce genetic material into another cell

  
• Plasmid features


  • Antibiotic resistance
  • Origin of replication
  • Promoter
  • Ribosome binding site
  • Multiple cloning sites
  • Start and stop codons
  • Gene encoding the protein of interest

  
• Cloning the gene


  1. Cloning from genomic DNA using PCR

  2. Synthetic DNA

  3. Ligating the gene into the plasmid

  4. Using ligation-independent cloning
• Bacterial transformation


  Making the cell passively permeable to DNA by incubation with divalent cations and heat shock
• Selection


  Growth on antibiotic-containing medium selects for cells containing the plasmid

  
• Lac operon


  Lactose transport and metabolism is under gene regulation, allowing effective digestion of lactose when glucose is unavailable

  
• Lac operon regulation


  1. In absence of lactose, the lac repressor binds to the operator and blocks transcription

  2. At low lactose and β-galactosidase, allolactose binds the repressor and induces a conformational change, allowing transcription

  3. At high β-galactosidase, the enzyme forms a tetramer that efficiently cleaves lactose

  
• IPTG


  A synthetic allolactose mimic that binds the lac repressor and induces a conformational change, without being metabolized

  
• T7 RNA polymerase


  • Faster than E. coli RNA polymerase, terminates transcription less frequently, highly selective for its own promoter
• Recombinant expression results in the overexpressed protein constituting a large proportion of the total protein in the cells
• Designer proteins


  Recombinant expression allows complete control over the gene sequence, enabling the biosynthesis of customized proteins

  
• Fusion proteins


  Two genes/sequences fused together, which can change properties like catalytic efficiency, solubility, thermostability, expression level, crystallisation, and purification
• Fusion-protein purification


  1. Proteins are often purified using affinity chromatography, with a purification tag like GST fused to the protein

  2. Proteases can be used to remove the purification tag, leaving behind a specific sequence

  
• Protease recognition sequences and cleavage sites


  • Thrombin: LVPR/GS
  • TEV: ENLYFQ/G
  • HRV-3C: LEVLFQ/GP
  • Enterokinase: DDDDK/
  • CleanCut: ELWSQ/X (X = any small amino acid)
  • Factor Xa: IEGR/

  
• It is very difficult to produce particular post-translationally modified proteoforms using recombinant expression
• Methods like mutagenesis, incorporation of unnatural amino acids, and ligation technologies can be used to produce customized proteins