Correct disulfide bond formation
Proteolytic cleavage of inactive precursor
See Fig 7.1 p241 for cleavage of preproinsulin
Glycosylation - addition of sugar residues
See Fig 7.2, 7.3 p. 242,243 in text
Modification of amino acids in protein
Phosphorylation // acetylation
Sulfation // fatty acid addition
2. (7pts) Answer the following questions regarding “error-prone” PCR:
a) What is “error-prone” PCR?
Use of the PCR under conditions that promote the insertion of an incorrect nucleoticde every few hundred or so nucleotides of the template. Used as a method of random mutagenesis.
b) What is one reaction condition of PCR that can enhance the error-rate of PCR?
in presence of Mn2+ // Unequal dNTP concentrations // Increased Mg2+ concentration
c) When would you use this method?
This method would be used when the specific amino acid to be altered isn’t known, but the general region to be mutated is known. Say, for example, the region of the active site or allosteric regulation, but not specific amino acids. Yields a library of mutations in a specific region of the gene of interest.
3. (6pts) Three of the four general categories of diagnostic methods are listed below. FOR TWO OF THESE METHODS, explain how each FAILS one of the three criteria (sensitivity, specificity and simplicity) that a diagnostic method should have:
1) Microscopic examination
2) In vitro culture and mouse inoculation
3) Detection of antibodies in serum
Microscopy – not sensitive
not specific because cannot discriminate between morphologically similar organisms
In vitro culture and mouse inoculation – not simple (uses animals, slow and expensive)
Detection of Ab in serum- not always specific
4. (4pts) One limitation often encountered when using recombinant proteins as therapeutics is a short half-life in the blood when delivered intravenously.
Describe ONE of the two strategies that were discussed in class that were used to stabilize recombinant Human Growth Hormone (rhGH).
Make a Fusion Protein
Fuse HGH sequence to stable protein - Human serum albumin Stabilize HGH in plasma, retain activity - Albutropin
Make a Fusion Protein
Attach the extracellular domain of the HGH receptor to the HGH protein Fusion proteins will dimerize (inactive), but in equilibrium with active monomer
5. (6pts) What are the two main drawbacks to using Antisense Oligonucleotides as a therapeutic strategy for treating a disease resulting from over-expression of a “normal” protein?
Oligonucleotides are susceptible to degradation
The high negative charge carried by the oligonucleotide makes it difficult to enter a cell – to cross the plasma membrane
6. (8pts) BIOtique Inc.’s Protein Expression Group is planning to product line to include a protein that is a dimer of two different polypeptide subunits, AB. As Director of this group, you have to instruct your technical staff of the requirements of the vector for the simultaneous expression of stoichiometric...