[10] Protein- and Immunoaffinity Purification of Multiprotein Complexes

We have not had any problems using this method to purify proteins that are lethal on overexpression, including Abplp and Duolp. Although cells are dying during the induction, significant amounts of recombinant protein are produced. Because some proteins are sensitive to longer (>4 hr) induction times, it may be important to test protein expression at different time points during the induction. The expression level varies from approximately 0.01 to 0.2 mg recombinant protein/ml high-speed supernatant, and all proteins tested have been soluble. The yield from these preparations is approximately 10% of the expressed protein; most of the loss is at the affinity purification step. We have not been successful in dialyzing out the putative inhibitor of binding to glutathione resins. We have successfully cleaved GST from GST-Aip lp bound to glutathione agarose using the thrombin site present in pEG(KT) and standard techniques, s but this approach has not worked for other more thrombin-sensitive proteins. To request plasmids or yeast strains described here, please contact Dr. Bruce Goode at [email protected].