No Ni-NTA binding--internal start codon of NdeI in pET vector? - (May/27/2008 )

Working with a 62 amino acid sequence subcloned into pET-14b at NdeI and BamHI sites--sequence verified, reading frame intact. There is a N-term 6x-His tag. With the tag, MW is 9.3kDa and 7.2kDa without.

I've attached photos of two gels (the second one literally got fried by a crappy apparatus! but still gives good info). Induction with 1mM IPTG yielded ~ 85% soluble protein, so I am purifying under native conditions. My protein is a bZIP DNA-binding protein, so it is a single alpha-helix with an intrinsically disordered N-terminus, therefore a His-tag is without a doubt fully exposed.

Buffers are typical Qiagen protocol:
Lysis/binding
50mM Sodium Phosphate (monobasic)
300mM NaCl
10mM Imidazole

Wash
50mM Sodium Phosphate (monobasic)
300mM NaCl
20mM Imidazole

Elution
50mM Sodium Phosphate (monobasic)
300mM NaCl
250mM Imidazole

The MW ladder indicators are on the left. I do not have a "peptide" or super low MW ladder, so I'm not sure if my protein band is at 9.3kDa or 7.2kDa (14.4kDa is the smallest on my ladder, these are 4-20% Tris-HCl SDS-PAGE gels). Also, I do not have an Anti-His antibody---- so please don't suggest I "try these first". The beads are bright blue, and are not reduced. Yes, the column was equilibrated before binding. I have tried both batch and column methods for binding, with equal negative results.

As we speak my protein is dialyzing out of the lysis buffer containing Imidazole and into plain 50mM Sodium Phosphate/300mM NaCl. I will attempt the purification again tomorrow and post pictures. However, I do not expect this to have any effect since 10mM Imidazole is an extremely low concentration, and should not inhibit binding of a well-exposed His-tag. I am wondering if anyone has experienced any internal translation initiation from the ATG in the NdeI restriction site after subcloning into pET vector. This may explain the lack of binding to Ni-NTA by yielding a truncated (7.2kDa) protein .

Hard to say but on the good gel it really looks rather like 9 than 7KD but again without good ladder it's hard to say just by estimation of the interspace between ladders

I had once a similar problem with a protein which bind weakly to the Ni-NTA column and was found in the wash, the best way to check proper sequence is to do a MS and see if your protein is really there.

Another option would be to try to purify on a NiNTA silica membrane spin column the adsorption is slightly different it might gives you a hint on the column quality, you can also try others ions columns like the Cobalt Talon agarose.

Did you try to purify it under denaturing conditions just to see itf they would be a role of secondary structure which would mask the His residues ?