BCH 5425 Molecular Biology and Biotechnology

Spring 1999

Tuesday Apr 27^th

NAME:_______________________

1. (10 points) For the following types of protein purification situations, indicate what you think would be the most important characteristic for an assay?

a. The contaminating proteins are closely related in molecular structure and function

b. The starting material contains the desired protein in trace amounts

c. The protein of interest is not very stable, and will loose activity over time

d. The protein of interest is environmentally toxic

(use the following chart for question #2 below)

2. The table below contains data from an initial fractionation step involving precipitation of proteins using polyethylene glycol (PEG). The numbers indicate the amount of total protein and desired protein remaining in the sample supernatant after addition of the indicated amount of PEG.

A. (10 points) From this data, calculate the specific activity and % yield of desired protein in both the super and pellet for each concentration of PEG.

B. (10 points) Based on this information what is the best purification you can achieve with the PEG precipitation? Under what situation might you decide to include this step in a purification scheme?

3. (10 points) Given the following information, explain what general type of ion-exchange resin, and containing what specific functional group, with you might use to purify the following proteins.

Proteins:

Experimental Buffer:

4. (5 points) A protein sample with a volume of 350ml contains NaCl at a concentration of 2.1 M. The sample is dialyzed versus 3.5 liters of 0.5 M NaCl. The resulting sample is then dialyzed versus 7.0 liters of distilled water. What is the final concentration of NaCl in the sample?

5. (10 points) The following figure represents an elution profile from an ion exchange column. The column was eluted with a linear gradient of NaCl from 0 to 2.0 M. The total volume of the salt gradient buffer was 140 mls. The fraction collector was set up to collect 2.0 ml fractions. The dead volume of the column and tubing leading up to the detector was observed to be 20 mls. Using this information, explain how you would set up a step elution to enable you to individually isolate the three eluted peaks.

6. (10 points) A gel filtration resin has the following technical specifications: it will exclude globular proteins with a molecular mass greater than 50,000 Da , and will completely include globular proteins with a molecular mass less than 6,000 Da. A sample containing the following proteins is loaded on the column:

7. (5 points) The following diagram shows two peaks that eluted from a chromatographic column. They are not completely resolved from each other and the individual contribution of each component to the overall profile is indicated.

A. For peak #2, which fractions would you pool to maximize yield?

B. For peak #2, which fractions would you pool to mazimize purity?

8. (10 points) Phage display techniques with bacteriophage M13 have made use of the major (gene VIII) coat protein to display variable peptide sequences. However, due to the large number of copies of this gene on the surface of the phage, binding interactions with a desired receptor can sometimes arise due to the combined effects of neighboring coat proteins. What are two approaches that have been utilized to minimize the number of displayed variable sequences (and thus minimize false positives due to neighboring interactions)?

9. (10 points) A yeast two-hybrid system, using the GAL4 system, is setup to test whether two proteins (protein "A" and protein "B") have a binding affinity for one another. The following plasmid constructs were made and gave the following levels of lacZ activity:

What conclusions do you draw from these data?

10. (10 points) Fill in the blanks for the following purification table:

11 (5 points EXTRA CREDIT) Answer the following questions with regard to the data in question #10

A. Do you think we have purified the protein?

B. Which step would you eliminate in an effort to improve the overall yield of the purification, and what might the resulting overall yield be?