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Rosetta-gami B Incubation Time


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#1 SeanWorkman

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Posted 09 July 2012 - 10:24 AM

Hey there,

I'm working on expressing a T. congolense protein in the Rosetta-gami B cells and while I am able to purify my target with no issues, I'm not entirely happy with the yield of protein I am seeing. I plan on setting up an autoinduction to run over the weekend, so incubating the cells for between 48 and 72 hours before harvesting, and hoping that my yield is better. How long is too long for this cell type, does anybody know?

Edited by SeanWorkman, 09 July 2012 - 10:24 AM.


#2 pDNA

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Posted 16 July 2012 - 11:32 AM

i would say 48 to 72 h is far to long for expression in E. coli ...it is not very reasonable to do it that way since the carbon source is depleted quite fast in batch mode ...and than your bugs start to starve and induce proteases that will degarde your protein.

If you want to increase yield it is more advisable to try to increase the total cell mass (e.g. by using richer medium like terrific broth or adding more glucose/glycerol whatever) and keep your culture induced for 2-4h (or even overnight). What i have seen so far is that the solubility gets worse with time for most of the proteins and overnight you end up with a huge amount of insoluble protein. Therefore, i always recommend short induction time but a high biomass concentration.

Best,
p




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