promlems in protein purification in native form - (Apr/29/2007 )

Hello !

I´m recently started to work with protein expression and I faced a promlem in studyin yhe function of my expressed enzyme: I cannot get any product out from the enzyme assays. I have expressed 40 kDa cytosolic homodimer protein in pQE-30 vector with a N-terminal His-tag in M15 E.coli cells. I have expressed the proteins at 22 degrees with 0,5 mM IPTG for 21 hours. Purification has been made according to QIAexpressionist under native conditions. I have studied the enzyme function assays (incubation of substrates with protein) both with crude protein extracts and purified proteins. No differences between tests and controls can be seen in the HPLC. In controls heat denaturated proteins were used.

I wonder what can be the promlem: enzymes not correctly folded or denaturated at some stage?

Can 22 degrees be still too high and proteins are not correctly folded? At 22 degrees very small amount of protein can be found with SDS-Page in supernatant but allmost all is still in the pellet = are insoluble. (At 30 degrees all proteins are in the pellet). Should I lower the temperature to for example 16 degrees or lower IPTG amount? Or is 21 hours too long time for expression? Or can it be that the bacterial host is not proper for those proteins and I need to change the host cells? For example due to rare codons? Thanks.

Sarita

You can run your sequence through the codon algorithm here http://gcua.schoedl.de/ . If you have too many rare codons, you will have issues getting any protein. Is the protein in the insoluble fraction full-length protein?
You can tweak the expression by temperature or IPTG concentration, but it's likely to only be a tweak, rather than a change from insoluble to soluble fraction.
Also, to make your day better, there are times when adding a His tag at the N-terminus can affect your protein. Have you considered a GST tag instead of His?

Well, sarita has the constructs with His Tag. Will be abit troublesome to change it to GST tag. You need additional enzyme to cleave the GST out.

As for the problem, I don't think it is the temperature problem. 22 Celcius is very low and should promote better protein folding. You can always try at lower level. And 21 hours I reckon it is abit too much. How about signal peptide? Is it with signal sequence or without? It plays an important role for better folding.