 |
 |
Weijun, There are literally hundreds of options you can pursue - obviously a comprehensive and detailed list of suggestions would not fit in a little email (in fact they don't all fit into a one-volume book). It would be helpful to narrow things down a bit - for starters, for one or two cases you may want to share with us exactly what you're trying to do. It would help to have the data pertaining to: the sequence of cDNA that you're using, where does the DNA come from, vector(s) you've tried, promoters, fusion proteins and tags attempted, strains of E. coli, media types, expression conditions, and so forth. Heterologous expression of proteins is not a guarrantee, and for each protein you try the solution may be different. All of the abovementioned variables and dozens more are important, and getting some of them right is not enough - typically you have to get nearly all of the variables right, and that can be a lot of work (and ultimately still may not be enough). As has been suggested, you can try other hosts - for instance Gram positive bacteria (Bacillus megaterium is an interesting host as are other bacilli, especially if your protein is secreted), other Gram negative bacteria (Pseudomonas for instance), yeasts (Pichia, Hansenula, Yarrowia, Klyveromyces), insect cells (Trichoplusia or Spodoptera - they're different!), mammalian cells (dozens of options there too), or cell-free expression. If you're truly desperate you can try expression in more esoteric hosts such as plant roots, algi, parasites (Leishmania), whole insect larvae, animals (milk), and so forth. Cheers, Artem -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Weijun Huang Sent: Friday, March 23, 2007 6:26 PM To: [log in to unmask] Subject: [ccp4bb] Inclusion body Dear All, I got enclusion body in many cases when I tried to express human proteins in E coli. I would like some suggestions on how to go about it. I would also like to try co-expression of GroEL/GroES or DnaJ/DnaK, and would like to know where to get the plasmids. Any help or comments would be appreciated. Weijun