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Please help me :Protein expression and purification


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#1 luoxuan

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Posted 25 June 2014 - 01:07 AM

Hi everyone, I am doing a protein expression and purification by E.Coli and some vector PET system,but there are many problems during my experiment.First,I should tell you something about my protein:400-500bp, cysteine rich protein and The repeat amino acid sequence such as AAAAAAA、LLLL 、TTT、RRR and so on.I use PET15b 、 PET28a and other PET Vectors cloned in BL21(DE3),However I can not find my Target protein through E.Coli culture through Ni-NTA purification by SDS-PAGE gel.Now I am very confused How and when I can get my target protein.Please help me!!!(ps:it is a member of LYNX,I am very thanks for you if you can give some articles about it)

#2 Jaber

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Posted 05 July 2014 - 07:32 PM

maybe you could look at the fermentation conditions especially the induction temperature cuz maybe the protein is expressed as an inclusion body which u can make sure about by SDS-PAGE of 1/OD samples during the fermentation 

i'm using pET28a with an induction temperature 28 C (induction with IPTG 1 mM)






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