20111118HW4 F11 ChE170

20111118HW4 F11 ChE170 - F11 ChE 170 Homework#4 Due...

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Unformatted text preview: F11 ChE 170 Homework #4 Due Tues Nov 29 1. Amgen’s biggest blockbuster drug Epogen (Erythropoitin) has recently gone off patent, prompting other companies to develop processes to produce generic (or biosimilar) versions. Use Genbank to identify the gene encoding human erythropoietin Genebank identifier (accession number) “NM_000799.2.” Using information in this file find and annotate the following: a. The first amino acid in the preprotein. b. The first amino acid in the mature protein (after signal peptide removal) c. The poly A tail 2. Use PDB and PyMol or Swiss PDB viewer to determine how many disulfides are present in EPO (use PDB file 1BUY). Provide a print ­out of your structure highlighting the disulfide bonds. 3. Design primers to amplify the gene encoding the mature EPO protein and insert into the pMAL vector (XmnI and EcoR1 sites) such that a minimum number of new (non native) amino acids are appended to the N ­terminus of EPO after fusion protein cleavage by the protease Factor Xa. Note: This problem is different from the in class example since the protein will be produced as a fusion to the C terminus of maltose binding protein. Be sure to maintain the correct reading frame. 4. The vector secretes protein into the periplasm. Why might this be beneficial for producing EPO? 5. After purifying the correctly folded protein from E. coli you determine that it increases red blood cell production weakly, but is not as potently as Amgen’s EPOGEN. What is the most likely explanation for this difference? 6. Question 10 ­6 (from your text) 7. Question 10 ­12 (from your text) 8. Question 10 ­13 (from your text) 9. Question 10 ­16 (from your text) ...
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