Bcbt 477 Proteomics, Advanced Protein Expression, and Chromatography Dr. Mazz Marry Open Book final exam – Due– Monday May 9th by 00pm Turn in only to the Chem Office- final deadline!!!!!!
The questions are weighted; see the point value for each question. Let the value of each question be the guide on the level of detail for each answer you give.
Use your notes and other resources for your answers. Simply using notes will not be sufficient for a grade of A or B.
Cutting and pasting notes or info from web pages is not acceptable and will result in a grade of F.
You have a wonderful new clone of a protein (Arsenal-are-the-greatest-soccer-team-in-the-world) that may solve all of the world’s hunger problems by converting paper (cellulose) into starch (HINT think or look up for how to assay a cellulose protein activity). You need to make a large amount of the protein.
What considerations do you need to make when planning the production of Arsenal-are-the-greatest-soccer-team-in-the-world? Be specific and thorough. Consider each possible step of the production from vector to expression level, host and purification.
What factors would you consider when choosing a particular host/expression system?
Even if you choose a fusion protein, it is not going to be purified in one step. What are some of additional factors that you need to be concerned with when purifying a protein?
What detection method (s) will you use to detect it?
PS your project manager does not like inclusion bodies so you need to consider that as well. You have all of the tools and vectors or plasmids you can buy or make. (200 points).
Explain what a fusion protein is and what affinity chromatography is. Outline the pros and cons of each of the various affinity tags. What are the differences in the fusion partners? What impact might the fusion partner have on a protein? What can be done about the fusion partner in the final protein product? What are the different mechanism/biochemistry of binding and elution for the fusion proteins? (200. points)
Prepare a full protocol for the chromatography of a 2 ml sample of a protein through a monoQ FPLC column. In addition to all of the standard parts of the protocol, you will need to load the sample, wash the non-binding sample through the column and elute the column with a 50 ml 0 to 250 mM NaCl gradient. Use a 10 mM Tris-Cl pH 7.4 buffer throughout the purification. (100 points).
Give a general description (not definition) of Proteomics. Explain why Proteomics is considered a very important field? (200 points).
Describe the various parts of Mass Spectroscopy. What are the key uses for mass spec? (100points)
Explain the ionization of protein samples for Mass Spectroscopy, and how you determine the molecular mass and the peptide sequence. (100 points).
What is transfection, how does the DNA cross the membrane and what is the difference between transient and stable transfection? (100 points).
Be Neat, Be precise, Be Complete. Avoid pronouns such as it, that, those and them, to ensure that you are clear and communicate your ideas well.
