try JM109(DE3) instead of BL21(DE3), combined with low temperature induction - for several proteins this has yielded us soluble protein instead of inclusion bodies. I think this is because JM109(DE3) grows a bit slower.
Mark J van Raaij [email protected] http://webspersoais.usc.es/mark.vanraaij/ researcherID: B-3678-2009 On 5 May 2010, at 14:54, Roger Rowlett wrote: > Several suggestions: > • Try tagging with a solubility-enhancing tag like GST or NusA. > • If using a pET vector system, try "leaky" expression. The T7 promoter > is not well-repressed (except in pLysS systems) and you can get low but very > significant levels of expression without induction. One protein system I have > worked with expresses very well under these conditions, and the low rate of > expression discourages inclusion body formation. > • You can attempt to clone tandem, triple, or quadruple repeats of your > gene behind a promoter. This works well for the expression of the alpha > subunit of Trp synthase. Supposedly, the gene repeats tie up transcription > machinery and slow down the rate of mRNA and protein production. Whatever. It > works. > • You have already tried lower temperature, and this often helps to > slow down overexpression and increase the soluble fraction of protein. You > could try going even lower in temperature and overexpressing longer. > Good luck! > > Cheers. > > > On 5/4/2010 5:11 PM, Buz Barstow wrote: >> Dear all, >> >> I am trying to purify a metalloprotein (a hydrogenase) using affinity >> chromatography. >> >> I have produced two tagged versions of the enzyme: one with an N-terminal 6x >> histidine affinity tag, and the other with a C-terminal 6x his-tag. The >> tagged proteins are both tied to an IPTG-inducible promoter. >> >> When trying to express and purify the N-terminal tagged protein, I have >> found that almost all of the expressed protein goes into inclusion bodies >> when the culture is grown at 37 or at 30 degrees C. When the culture is >> grown at 20 degrees C, a small amount of protein can be found in the cell >> extract. >> >> Unfortunately, as the enzyme has several oxygen-sensitive metal clusters, we >> do not believe that the protein can be refolded from the inclusion bodies. >> >> Could you offer some advice on how to express and purify this protein and >> reduce the quantity of protein found in inclusion bodies? >> >> Thanks! and all the best, >> >> --Buz >> >> > -- > Roger S. Rowlett > Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: [email protected]
