Slide show

1. Cloning strategies - a brief word (or two)

Resource reading

• Deduction of codon sequence from protein sequence
  • Edman degradation
  • Backtranslate from protein sequence

    Example: Met Asp Pro Leu Trp

    AUG GAC CCG CTG TGG T A A T T C C T G T A How many-fold degenerate is this?

  • Prepare degenerate PCR oligos
  • Isolate sequence(s) and characterize
• Antibody preparation against a purified protein, and screening of an expression library
• Functional complementation
• Physical mapping and analysis of candidate gene
• Enhancing procedures
  • Size fractionation of RNA
  • Selection from pools
  • Hybrid select translation/Hybrid arrest

2. Engineering considerations

Resource reading

Domain shuffling

• Example of IFN-alpha genes (Fig 10.1)
• Alternate methods - what we've learned in past weeks about mutagenesis

General mutagenesis for augmentation or constraint

• hGH binding characteristics with GH receptor or prolactin receptor

Resource reading

• Optimization of expression in different hosts
  • yield
  • activity

3. Enzymes

• DNase I
• Alginate lyase
  • Screening method

4. Antibodies

• Passive immunity
  • Balto
  • Rhogam
• Antibody structure
• Chemically-linked monoclonal antibodies
  • drug concentration problem
  • activation of a prodrug
  • tPA
• Humanization of antibodies
• Transgenics
• ScFv and phage display

Dr. Stan Metzenberg Department of Biology California State University Northridge