Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins

[Roger Rowlett]

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: rrowl...@colgate.edu