It is unlikely that a his-tag would be a source of issues regarding the formation of inclusion bodies for a recombinant protein. Not impossible but unlikely. It could be the case in your particular situation but it is far more likely that your particular human protein does not do well expressed in a prokaryotic system. If you express without a tag do you still get inclusion bodies? If that is the case then going for a different tag or altering the position N-term/C-term may not be of any practical benefit. You could attempt another host system (yeast/insect/mammalian) or you could attempt to refold from inclusion body. It really depends on your particular protein and the time and resources you have available. And if you are concerned with long term solubility with the tag, you can of course always remove it post expression.

Well, solubility can not be directly correlated with the fusion tags, your protein of interest may tend to form insoluble or aggregates due to several reasons in the given buffer.As far as I know, 6X His may hinder the protein dynamics(if your doing NMR or other things) but not promote aggregation in most cases.I suggest you to test with different buffer/pH/ionic strength/purity/glycerol/ and tags!

Good luck.

If recombinant protein solubility is an issue, there is a hyperdilution method that you can try that worked for me. It involves slowly adding the guanidine dissolved inclusion bodies into a refolding buffer: 0.1M Tris pH8, 2mM EDTA, pH8, 0.4M L-arginine hydrochloride, 0.5mM oxidised glutathione, 5mM reduced glutathione, leave at 4 °C overnight then concentrated and dialyze. Worth a try!

It is unlikely that a his-tag would be a source of issues regarding the formation of inclusion bodies for a recombinant protein. Not impossible but unlikely. It could be the case in your particular situation but it is far more likely that your particular human protein does not do well expressed in a prokaryotic system. If you express without a tag do you still get inclusion bodies? If that is the case then going for a different tag or altering the position N-term/C-term may not be of any practical benefit. You could attempt another host system (yeast/insect/mammalian) or you could attempt to refold from inclusion body. It really depends on your particular protein and the time and resources you have available. And if you are concerned with long term solubility with the tag, you can of course always remove it post expression.

I work for a high-throughput structural genomics facility as part of a PSI:biology center which ships about 80 to 100 purified proteins a month. For some classes of proteins, such as some Enolases, the His-tag (both N and C term) has caused a decrease in solubility. If using a solubility enhancer, like a SUMO fusion, isn't possible, then you may want to work tagless. The His tag becoming a problem is rare, so it still may be worth trying the tag on both termini.

Recombinant protein solubility depends on lots of factors. It, of course, depends on tag, but it is not often to change solubility very well by replacing a tag. You might want to try various culture conditions, which also change solubility of recombinant proteins. There are articles showing that heat shock is useful and that lactose instead of IPTG help to get soluble proteins (I don't have those articles,though). Even if you get lots of insoluble proteins, some amount of soluble protein could be obtained. See if you can get enough protein in the soluble fraction. Some detergents also help to solubilize proteins, you may already consider that, though.

Another suggestion would be to clone your protein cDNA into a vector such as pMAL which makes a maltose binding protein (MBP)-fusion protein. MBP has been shown to increase recombinant protein solubility. Purification is done by using an amylose resin (only made by New England BioLabs) which binds the MBP. The construct we use also has a 6X-his tag which gives the option to purify using Ni2+ columns. Another nice feature of this construct is that it has a tobacco etch virus (TEV) cleavage site to allow removal of the MBP protein after purification, if desired. There are different varieties and ways to construct the His-MBP fusion (placement of the his tag in between the MBP and your protein, for example, if you want to retain the his tag, but cleave off the MBP). I would check out addgene.org as an inexpensive way to try out different plasmids.

You can also try inducing at lower temps which has also been helpful in increasing protein solubility. For example, you can try inducing at 22-25 degrees Celsius overnight. I would try a range of temps to see what is optimal. Although we haven't tried it, others that I've talked to say that auto-induction (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2747370) works well for difficult proteins.

@ Lindsay, I am interested in your refolding protocol, from where did you get it? is developed by you or it is published?

Thank you,