Rapid purification of recombinant proteins fused to chicken avidin

A novel expression vector (pAVEX16C) has been constructed that directs the synthesis of desired polypetides as fusions with the C terminus of chicken egg-white avidin (Avd). With this and a commercial GST gene (encoding glutathione S-transferase) fusion vector (pGEX-3X, Pharmacia), we produced Avd as fusions C- and N-terminally linked to GST in Escherichia coli. By using the Avd tail and a simple affinity purification protocol, including biotin-agarose, we were able to obtain 1-2 mu g/ml of highly purified AVd::GST and GST::AVd from crude bacterial lysates. The produced proteins were, to a great extent, in soluble fraction when the cells were grown at 22 degrees C and disrupted with a detergent, N-laurylsarcosine. The fusion proteins could also be affinity-purified with the GST tail using glutathione-Sepharose 4B, but the yield:of GST::Avd was significantly lower than when using the Avd tail. Our results therefore indicate that it is possible to produce, in E. coli, biologically active fusion proteins consisting of Avd C- or N-terminally linked with the desired protein which then can easily be purified by a simple affinity chromatography procedure.