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Improving expression of recomb proteins in baculovirus

Sf21 insect expression

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#1 JenH

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Posted 05 September 2011 - 07:45 AM

Hi

My work involves expressing a number of different recom. proteins in insect cells and then purifiying by IMAC, GST or FLAG. Some proteins express/purify nicely, others not so well. I have been tasked with trying to improve our expression conditions and I wondered if anyone could point me in the direction of some good literature - I am thinking along the lines of alternative promotors, additives to the media, possible use of chaperones (maybe?) or anything that isn't just 'add more virus' or "change length of expression time". I'd be gratreful for any comments or a good push in the right direction as this is not my field of expertise.

thankyou

#2 protolder

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Posted 05 September 2011 - 09:51 PM

Hola, you give short information, but I will try to help you. Firstly the system is very efficient, you have known , but few times fail, I have expressed lot of protein but one day you aren´t able to express some protein in particular and see that other groups or dealers neither, is the case of PI3K beta that seems to be unstable or difficult to express by any cause. Well the first clue is to have the producer clone well isolated and characterized, if you use bac to bac, you will select it in bacteria or if you use analogous recombination with commercial digested DNA there is a little % of undigested DNA that could make to lost expression with passages, so you have to select recombinant viral clones. Easily you have to have expression extending the time to collect cells untill infection symptoms were seen, but the correct way is titering the passages and making a time course experiment with different multiplicities of infection (MOI), moreover if you need important amount of protein and infect a high number of cells. If you use serumfree media or specific cells as Mimic in my knowledge higher MOI have to be used. Buena suerte





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