Hola I don´t know if you makes digestion when the fusion protein is bound to resin or after elution. For me It´s better elute the fusion protein and made digestion at low concentration and pH as far as possible of isoelectric point of your protein, (always into the range of activity of protease). After the digestion pass the preparation across the column and recover free peptide from flowthrought. For concentrate it, an ultrafiltration membrane of 3Kd or a ammonium sulphate precipitation followed from a gel filtration could helps you to have your peptide. If this method continues giving aggregates don´t worry to use Urea in all the media after digestion. Buena suerte
After some months of inactivity I moved from protein expression and purification in Oncology to protein expression at high scale in fermenters in the area of nitrogen fixation. This is my first contribution asking for some aid.
Some of the strains , which I use are grown in aerobic conditions until ammonia depletion and the induction of the nitrogen fixation complex take place. In some strains this induction is better in anaerobiosis, so after grow in normal aireation conditions, the sole gas introduced in the fermenter is nitrogen. At the beginning of anaerobiosis the dissolved oxigen drops to zero but few minutes after it¨s observed that dissolved oxigen starts to increase to 10% as maximun depending of conditions. After the maximun it could be mantained or decrease in some cases. At the begining, I thougth that the probe was bad calibrated, but I check good calibration in some of posterior processes and this release continues. Moreover, after harvesting I fill the fermenter with water and passing nitrogen, the dissolved oxigen is zero. I have search in publications about this phenomenon and few information in this field has been published. If it´s difficult find well described fermentation procedures, in anaerobiosis it seems impossible. Does anybody have any explanation for this process?
Hola,I had similar problems with Mimic Sf9 cells , but the cells arrived dead.In your case, in which cells are living, I would seed in a B25 flask and let form monolayer in sf900. After, pass to a B75 scraping monolayer or pumping medium with a pipette, and when the density was enough to seed in suspension I will do it. This cells are able to give 5x10e6 cells/ml. Sorry by the delayed answer. Buena suerte