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Expression of insoluble protein

media E. coli strain Solubility

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4 replies to this topic

#1 hRNA

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Posted 03 May 2012 - 05:37 AM

Hi All
I am facing a serious problem in expression of a protein in soluble fraction
I have a full-length gene (approx. 1.6kb) cloned in pExp5 TOPO/ CT vector.
Sequencing of the clones revealed that the gene is in-frame. I have expressed it in Rosetta-BL21(DE3)pLys-S cells at 18 C in an autoinduction media. I could see the clear expression in lysed cell pellate (~64KDa) when SDS-PAGE was done, whereas the final elute lane in the gel was blank. with several trials of media, incubation time and few other practices I was unable to get protein in the soluble fraction.
With this, I have thought of cloning the gene in pET32(Posted Image. But in this case I am unble to get the clone.
Can anybody help me to identify suitable vector for cloning the gene which will give atleast partial solubility to the protein.

#2 pDNA

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Posted 08 May 2012 - 12:55 PM

what expression strength did you use?
maybe your gene is toxic to the cells when soluble?

pET-based expression is often to strong ...you could try weaker expression e.g. using pBAD vectors (inducible with arabinose).

Regards,
p

#3 hRNA

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Posted 08 May 2012 - 08:29 PM

thanks for ur reply
i had gone throught the pBAD vector details. I will now try to clone it in the same.
Thanks once again for this information.
i would also like to know whether u have any experience with pBAD vectors?
do they work well in terms of cloning and expression?

#4 pDNA

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Posted 08 May 2012 - 11:35 PM

you should use a strain with deletion in the araD gene for optimal performance (DH10B, TOP10) ...then arabinose acts in a way similar to IPTG (no degradation of inductor) otherwise in "normal" strains it is metabolized. Additionally bear in mind that arabinose is only taken up into the cells when glucose is absent ...therefore use a different C-source than glucose (e.g. glycerol or LB).

Best,
p

#5 hRNA

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Posted 09 May 2012 - 08:59 PM

Hi
thanks once again in helping me out with this information.

Best





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