His-Tag:Single column prep?

[Stephan Witte]

In article <1995May15.212901.16374 at rs6000.cmp.ilstu.edu>,
ajotsuka at rs6000.cmp.ilstu.edu (Anthony Otsuka) wrote:
> 
> We have been using pRSET vectors (Invitrogen) for expressing
> proteins.  During purification over nickel chelating columns,
> we see a considerable amount of background protein binding
> (other metal-binding proteins?).  Has anyone been able to
> achieve >90% purity by using the single column?  Any advice
> would be appreciated.  Thanks, A. Otsuka

Hi,
I«ve been working for now more than 2 years on expressing some human
ribosomalproteins using the pRSET vectors 
(Expression is done in BL21 or BLR from Novagen). Because my proteins are
expressed in inclusion-bodies, i«m doing the purification under denaturing
conditions followed by a renaturation step.
Usually I get more than 95% purity on a single column. 
Sometimes, especially on purifying whole bacteria lysates  on the resin, my
preparations contain a impurity, which appears at about 29 kD in SDS-PAGE.
I can find this impurity even in control experiments using bacteria with
pRSET wihout insert or bacteria without plasmid. This suggests that this
protein is a e. coli protein with some affinity to the resin.
When I first isolate the Inclusion Bodies and then do the affinity
chromatography I usually achieve purities of more than 98%.
Some hints for avoiding a general protein background:
						1. stringent washing of the resin after loading to the beads:
						(at least 20-50 times the beads volume) with 20-60 mM imidazole
(native purification) or 8M urea pH 6.0-6.3.
						(You have to find out the best washing conditions for your protein by
your own) 
						Best results you get with monitoring the washing steps with SDS-PAGE
or UV-detection (most convenient)

						2. Use less of the Ni-NTA-agarose:
						1 ml of of the beads are sufficient for binding 5-15 mg of His-tagged
proteins. 
			
						3. There is a very good manual available from QIAGEN Inc. called "The
QIAexpressionist"

I hope this helps, contact me for detailed information or just discussion.
Im interested on every stuff concerning the purification of His-tagged
proteins.

Stephan


         																			 ---Sorry for my horrible german english!?!---	


Stephan Witte
Inst. of Immunology
University of Constance
stephan.witte at uni-konstanz.de