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pQE vector protein expression


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#1 hothot

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Posted 02 April 2009 - 01:41 PM

I was having difficulty to express one protein with pQE vector. I insert the gene into pQE vector and sequenced it. Everything seems to be all right. But I just can not see any protein expressed when IPTG was added. I even used western blot to detect if there's any HIS tagged protein but turned out to be none.

Could anybody here give me some suggestions. I was so desperate to find out why I can not induce it.

#2 T C

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Posted 02 April 2009 - 06:30 PM

Hey,

It happens...the protein could be toxic to the cells or could be utilizing rare codon (thus you use codon surplus Rp or RIL strains)

Try in RP if GC rich and RIL if AT rich and see if it expresses.

Best,
TC

I was having difficulty to express one protein with pQE vector. I insert the gene into pQE vector and sequenced it. Everything seems to be all right. But I just can not see any protein expressed when IPTG was added. I even used western blot to detect if there's any HIS tagged protein but turned out to be none.

Could anybody here give me some suggestions. I was so desperate to find out why I can not induce it.



#3 hothot

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Posted 03 April 2009 - 01:22 PM

It does have rare codons, and I tried codonplus, and I tried some others, but it did not work.

Plus, the protein I am working on is a bacterial protein, I don't think it will be very toxic.
I was really frustrated.

Hey,

It happens...the protein could be toxic to the cells or could be utilizing rare codon (thus you use codon surplus Rp or RIL strains)

Try in RP if GC rich and RIL if AT rich and see if it expresses.

Best,
TC

I was having difficulty to express one protein with pQE vector. I insert the gene into pQE vector and sequenced it. Everything seems to be all right. But I just can not see any protein expressed when IPTG was added. I even used western blot to detect if there's any HIS tagged protein but turned out to be none.

Could anybody here give me some suggestions. I was so desperate to find out why I can not induce it.



#4 T C

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Posted 03 April 2009 - 07:47 PM

Hey,

It happens, try changing the host. Its best to overexpress it in the organism that it belongs to or some related organism. For instance Mtb protein will express in Msmeg but the toold must be well defined or you need to develop them.

Best,
TC

It does have rare codons, and I tried codonplus, and I tried some others, but it did not work.

Plus, the protein I am working on is a bacterial protein, I don't think it will be very toxic.
I was really frustrated.

Hey,

It happens...the protein could be toxic to the cells or could be utilizing rare codon (thus you use codon surplus Rp or RIL strains)

Try in RP if GC rich and RIL if AT rich and see if it expresses.

Best,
TC

I was having difficulty to express one protein with pQE vector. I insert the gene into pQE vector and sequenced it. Everything seems to be all right. But I just can not see any protein expressed when IPTG was added. I even used western blot to detect if there's any HIS tagged protein but turned out to be none.

Could anybody here give me some suggestions. I was so desperate to find out why I can not induce it.






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