Posted 14 September 2011 - 10:27 PM
Hola, periplasmic expression vectors are designed to have a clean periplasmic protein fraction at the first purification step. If you sonicate you will have two protein species: the processed periplasmic one and the non processed cytoplasmic , wich has a Nt signal peptide. If your construction has the histidine tag after the Nt SP and you have your expression induced could occurs that you see a lot of protein that not bound the resin. if your tag is Ct i haven´t explanation. But think a thing more, secretion systems in all organisms have a handycap you are increasing your recombinant protein expression, that has to be processed by the normal processing machinery wich is easyly saturated, so it ´s better a light induction in a long period before harvesting.
Look for the osmotic shock method for your purification and one more aspect, I think that the amount of detergent in your buffer is a bit high. Buena suerte