338 #11

• Mutagenesis


  Changing particular residues in a protein

  
• Mutagenesis


  • Mimic a mutation found in nature; disease state
  • Probe importance of a residue, e.g. in an enzyme, in an interface or for interaction, a protein's mechanism etc.
  • Mutagenesis using UV radiation or mutagenic chemicals is not desirable as cannot control the site of mutation, or what the mutation will be

  
• Site-directed mutagenesis


  Site-specific mutagenesis that gives the power to formulate and test specific hypotheses about protein structure

  
• Site-directed mutagenesis


  1. Use DNA polymerases to extend oligonucleotide primers on a plasmid template

  2. Requires synthetic oligonucleotide primer that has the mutant DNA sequence, and a template plasmid containing the gene

  
• Mutagenesis primers


  • Design primers that overlap the bases to be mutated
  • These primers contain the 'new' sequence of bases

  
• PCR (Polymerase Chain Reaction)


  1. Amplify the plasmid containing the gene

  2. The products will contain the substituted bases

  
• PCR


  • Ingredients: DNA polymerase, dNTPs, primers containing the new sequence, template plasmid
  • Typical PCR cycling protocol: Initiation, Denaturation, Annealing, Extension/elongation, Final elongation

  
• Bacterial transformation of new plasmid


  1. PCR mixture contains the old plasmid and many copies of the new mutated plasmids

  2. Treat with restriction endonuclease to remove the original plasmid

  
• Determining successful mutagenesis


  1. Sequence the plasmids to check if the mutagenesis was successful

  2. Transform the successfully mutated plasmids into bacteria

  3. Express the variant protein

  
• Designing primers


  • Plasmid DNA is double-stranded
  • Sense strand can be used to read the expected protein sequence; anti-sense in the opposite strand
  • DNA polymerases add nucleotides to the 3'-end of a DNA strand, so primers are designed in the 5' to 3' direction
  • Primers will be the reverse complement of each other

  
• Designing mutagenic primers


  • Mutagenic primers will contain the new bases that are not complementary to the plasmid DNA sequence
  • Regions on either side of the mutated bases will be complementary to the plasmid DNA sequence

  
• Beyond site-directed mutagenesis


  • Site-specifically change any (or multiple) amino acids in a protein
  • Incorporate unnatural amino acids by hijacking and modifying the cellular machinery

  
• Aminoacyl-tRNA synthetases


  • Attach the amino acid (or alternative) on to the tRNA
  • Have specificity for a tRNA molecule, and the amino acid
• Codon usage in the genetic code


  • There are no free codons
  • Some codons are rarer than others

  
• Discovery of the amber stop codon


  • Phage can have mutations creating an amber stop codon
  • Some E. coli strains have a pre-existing amber suppressor tRNA mutation that can "read through" the amber mutant
• Naming of the amber stop codon


  Named after Harris Bernstein's mother as a joke
• Amber stop codons/suppression


  • Discovery of 'tRNA systems' for amber stop codons means a method to incorporate unnatural amino acids
  • Key requirements: unnatural amino acid, unused codon, tRNA that recognises the codon, tRNA synthetase that recognises only that tRNA and amino acid, tRNA and synthetase must be functionally compatible with the translation apparatus

  
• Using the amber stop codon system


  1. Site-directed mutagenesis to incorporate the amber codon at the particular site(s) in the protein

  2. Directed evolution of the synthetase to change what amino acid the tRNA is charged with

  
• Features of the expanded genetic code


  • One or more specific codons can be re-allocated to encode an amino acid that is not among the 20 common ones
  • Can genetically direct unnatural amino acid to any chosen site in the protein of interest
  • Different or new protein functionality can be ribosomally incorporated into proteins
  • Scalability, limitations on protein size?
  • What does this mean in terms of fidelity and efficiency?
  • How does this compare to post-translational modification?
  • Allows to site-specifically label in vivo

  
• Applications of incorporating un-natural amino acids


  • Probing protein structure and function
  • Probing the role of post-translational modifications
  • Identifying and regulating protein activity
  • Selective destruction of selected cellular components