Extra bands after protein expression/purification - (Mar/09/2012 )

I have previously expressed two His-tagged proteins and they show no additional bands after purification. I created a fusion His-tagged protein by combining these two proteins and then purified it using a Nickel column. However I observe bands at lower molecular weights but the predominant band is the correct band. The figure i attached has lane 2: the fusion protein, lane 3: protein 1, lane 4: protein 2. How can I get rid of these incorrect bands in lane 2?

I used 0.5 mM IPTG to induce a 1.0 OD culture (750ml)

Would it help to lower the IPTG and maybe do a 0.6 OD culture? I induce at 20C overnight

Any advise would be greatly appreciated!

Thanks

lower molecular weight possibly means cleavage by proteases ...do you use protease inhibitor?

Regards,
p

pDNA on Sat Mar 10 08:34:12 2012 said:

I do not use protease inhibitor because the protein of interest has protease activity and I do not want to disrupt that. Also, this protein cannot self-cleave since the sites needed are disrupted

the protein can not self-cleave ...but can be cleaved by E. coli proteases (and since you observed bands at lower molecular weights this seems very likely) ...try to work on 4°C after cell harvest ...and maybe there is a protease available that does not inhibit your protease but does inhibit other proteases?

If you still suffer from degradation you will have to purify your protein after His-Trapping by size exclusion to get rid of the degradation products.

Regards,
p

Thanks pDNA. I am repeating the experiment and will work on ice after I pellet the cells. I am coupling this with using a 0.6 OD and lower IPTG concentrations.

I did run the sample through a millipore 30,000 MW filter and it appeared to not remove any contaminants! the 30,000 MW is well below my protein weight ~70,000 and should have removed most contaminating bands. After running on a gel, the filtrate and the fraction that was stopped by the filter look identical. Another student in my lab told me the filters were unreliable and I have to agree. Has anyone used these millipore microcon filters?