hello Andy, i have different questions about your problem :
Is the hetero-dimer co-expressed ? Or the two partners are express separately ? Have you controlled "where" are you proteins after the lysis, because if you have many of your proteins in the pellet, it's not a good sign for the folding state. If this is the case, you have different solution : change the growing conditions, change the buffer for the lysis step... If you have a low amount of protein in the first step of your purification process, you have to control at each step which one is the more limiting. Because, it's maybe not really necessary to do two affinity column. You can try different condition for the purification and increase the separation. This may allow you to skip one step and go directly to the size exclusion column. Sometimes the problems with the desalting column is the high speed of the variation. This high speed is potential source of osmotic stress for the protein. Need you really to reduce the amount of salt ? If it's absolutely required, you can try to do by dialysis, but by multiple steps. Another question, as soon as you can, control the good state of your protein ( for example with Circular dichroisme or DLS). Hope to help you. Nicolas ________________________________________ De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Anindito Sen [andysen.to...@gmail.com] Envoyé : jeudi 30 janvier 2014 14:17 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Protein Purification Problem Dear All, This may be slightly off-the-track question but your feedback will be very much appreciated. The situation is- I obtain a very low amount of the protein of my interest (a hetro-dimer) from the construct I am using (only 8% of the total amount of protein obtained is the protein of my interest). After 2 column purifications (Ni-NTA and St) the concentration of the protein is around 0.24 mg/ml (volume- ~1.0 ml) from a litre of bacterial culture and in ~300 mM NaCl present in the elution buffer. To reduce the high amount of salt I have I use a desalting column which, further lowers the protein concentration significantly. I need atleast 1.0mg/ml of protein concentration and to the amount of ~200 microlts for further experiments. As the last resort I try to use high amount of bacterial culture (~6lts) to scale up the yield and use centricon to concentrate the protein at various stages. I am partially successful to obtain 0.56mg/ml of protein concentration and up to 50 microlts of it. Another problem is that the protein is notoriously prone to aggregation (> 1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce the high salt concentration has failed miserably. Please do send your feedback. Thanks and Best Wishes Andy Dr. Anindito Sen (Ph.D) Department of Cell Biology & Anatomy Graduate School of Medicine University of Tokyo Tel & fax: +81-3-5841-3339
