Friday, June 22, 2007
Question 17 - Protein purification
Affinity-tag protein purification has four major steps:
(1) Development of the vector and expression system
(2) Obtaining soluble protein lysate,
(3) Isolating your protein and
(4) Possibly removing your affinity-tag.
Take this into consideration as you purify the hypothetical protein Trouble-maker (TrbM). TrbM is a 35KDa protein of bacterial origin. You first attempt to purify the protein using an affinity-tag. Unfortunately, you run into several problems (listed below).
For each problem, list and explain ONE solution for the problem. BE BRIEF! Only 1-2 sentences per resolution.
A. The first affinity-tag you try is an N-terminal His6 tag. Because TrbM is bacterial in origin, you decide to use an E. coli expression system. When using the pET system, your cell cultures stop growing after adding IPTG to 0.1mM. A small scale test indicates that the cells did not produce your protein. How could you increase protein expression?
(Some clarification: the pET system is an expression vector with the lacI gene which codes for the lac repressor protein. IPTG is an analogue of lactose.)
B. You now have good expression. You test protein solubility on a small scale. You use lysozyme to lyse the cells. You decide there is enough soluble protein to continue with a large scale culture. After lysing with lysozyme and sonication, you check for soluble protein (see picture below). Consider what problems you may be encountering. How would you increase the amount of soluble protein of interest?
Clarification: “supernatant from lysis” means that the lysate was centrifuged to remove heavy particles/debris. If the desired protein is soluble, it’s usually found in the supernatant.
(Hints: identify which lane is different. What does the difference imply? What else in the brief description above could cause this? What is not mentioned in the brief description that could cause this?)
this is why im gg to fail my h3 cos i have no idea what the above means.
anyway, my answer to the last question would be "CONTAMINATION!"
i just realised i forgot to blog abt enCOre.
i think it was great! except for the part where i had to keep flipping my score cos i cldnt find the correct page for tian shan zhi chun.
thanks alicia szemin michelle melinda and their other halves for coming down to support, as well as the sc2 ppl :wudi evon simran tzening
thanks for all the chocs (dom) and the flowers!!!!
im sure I'll get occasional bouts of COws, aww my sexy zhongruan. hahaha
thank you co ppl for making co so enjoyable, and of course, the pretty zhongruan and tanbo ppl! =)))
rain on your parade
1:49 AM
