GEN #14 Recombinant Protein Methods

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HUSOP2014
ID:
139064
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GEN #14 Recombinant Protein Methods
Updated:
2012-03-02 08:23:08
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HUSOP GEN EXAM2
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GEN #14 Recombinant Protein Methods
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  1. Banting & Best isolated insulin enriched fluid from _____ of dogs whose pancreatic duct had been _______.
    • islets
    • ligated
  2. Insulin Protein is a ________ primary gene product (INS gene).
    Proinsulin
  3. Insulin Protein is processed by ________ and _________to remove the __________ portion.
    • prohormone convertase-1 & -2
    • carboxypeptidase
    • C-peptide
  4. The final insulin protein has two chains _____ & _______ linked by ________bridge
    • A-21 aa
    • B-30 aa
    • disulfide
  5. Insulin protein is highly conserved with _____ difference for porcine insulin and ______ difference for bovine
    • 1aa
    • 3aa
  6. In addition to a disulfide bridge the A & B chains are also linked by a _____ _______
    peptide bond
  7. The expression vector to use will depend on
    • the size of the cDNA to be inserted
    • the host cell into which the vector will be introduced
    • the need for production of a fusion protein
  8. Bacterial cells preferred for recombinant proteins because
    • E. Coli genetics well understood
    • Large amounts of protein expressed
    • Fermentation technology advanced
  9. Yeast or COS cells are
    • Simplest of eukaryotic cells
    • Can grow in large bioreactors
  10. COS cells have to grow on
    solid support
  11. The two major problems with E. Coli production of proteins
    • Inclusion bodies
    • Solubility of proteins may be low
  12. Low solubility of proteins is why the protein may need to be ______ to a _______ __________ that can be removed later in the processing.
    • fused
    • soluble protein
  13. The recombinant protein needs to be purified
    from all ________ ____________
    cellular components
  14. The recombinant protein purity should be
    >99%
  15. The recombinant protein should be kept in its
    native conformation
  16. The ______ of the recombinant protein should be maintained
    solubility
  17. Protein is released form the cells by physically or chemically _________ _______ the cells
    breaking open
  18. Stabilize the proteins and protect from ________
    proteases
  19. __________ such as _________ _________ ___________ or ________ _________ may be needed to keep the protein soluble during purification
    • detergents
    • sodium dodecyl sulfate
    • sodium desoxycholate
  20. _________ selectively removes large molecules from a mixture
    Precipitation
  21. Precipitation may lead to ________ ___________
    protein denaturation
  22. Commonly used separating agents include ________ ______ and __________ ________
    • ammonium sulfate
    • sodium chloride
  23. _______ is done on a molecular sieve sized basis
    Filtration
  24. ____________ is the technique of choice for separating proteins
    chromatography
  25. Chromatography can separate molecules based on
    • molecular weight [size exclusion]
    • ionic charge [ion exchange]
    • hydrophobicity interaction
    • specific functionality (affinity chromatography)
  26. The AA sequence should not be _________
    altered
  27. ___________ is used to ensure the protein is in the correct 3D conformation.
    crystallography

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