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difficulty to clone and/or express proteases. Toxicity?

protease dh5 alpha induction cloning

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#1 monicalvi

monicalvi

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Posted 19 April 2013 - 12:10 PM

Hello.

I've been trying to clone and express a family of bacterial proteases. The protease is expressed as a pre-pro-protease, and the pre-pro domains are thought to the cleaved during maturation.

I firstly cloned the full lenght genes. I had some difficulties in cloning, but after some time I succeded. But the proteins turned out to be impossible to express in the strains (BL21-SI, C43, DE3, Star pLyS...) and protocols I used (different inductor concentrations, temperatures, induction times). I believe the proteins were toxic to the bacteria, as for example, BL21-SI did not grow after transformation.

I decided to clone the genes without the pre and pro domains. It seems that I can only cloneor express when the gene product is not active.

Is it possible that the gene is toxic to the bacteria even before induction? And could this happen in DH5 alpha using a T7 promoter vector???

What could I do to overcome this problems? Any ideas?

If more information is necessary to solve my problem, feel free to ask me.

Thank you sooooooo much in advance.





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