 |
 |
On 02/15/2016 09:40 AM, Elias Fernandez wrote: > Dear colleagues, > > Thanks for the many approaches to address this problem. I neglected to > mention that > > 1. We have an N-terminal His6 tag to the MBP-fusion and first purify > the complex with Ni-NTA. Thus, it's likely that either the fusion protein is > getting cleaved or it's a transcription/translation problem. > > 2. We get approx. 50:50 MBP and fusion but the total MBP + MBP-fusion > are very low. > > 3. We can partially separate the two by size exclusion. > > 4. We've not had much luck with other tags, such as SUMO and GST. > > Best regards, > > Elias Hello, if desperate you can try intein-based system (NEB sells it as 'IMAPACT system') either alone or combined with his-tag (ie His-prot-intein construct) or even MBP (ie His-MBP-prot-intein). The final yield might be crappy (especially when both MBP & intein tags are used) but whatever gets through is nearly sure the be the expected protein due to affinity tags present at both ends of the construct. Conveniently, intein can cleave itself off C-terminal end of the construct leaving no trace of itself. lukasz -- ------------------------------------------------------------------------- Lukasz Salwinski PHONE: 310-825-1402 UCLA-DOE Institute FAX: 310-206-3914 UCLA, Los Angeles EMAIL: [log in to unmask] -------------------------------------------------------------------------