Purification of dual-tagged intact recombinant proteins

Large-scale purification of recombinant proteins has been used extensively to assist numerous protein studies, including investigation of function, su bstrate identification and protein-protein interaction of low abundance pro teins. Genetic fusion of affinity tags to these proteins has also been wide ly used for ease of purification by affinity chromatography. However, this technique sometimes yields unstable and degraded protein products limiting its application. In this study, we show a facile and straightforward method of dual-tagged recombinant protein purification that eliminates contaminat ion by degraded protein products. A 6His-containing BamHI-HindIII fragment from pQE12 was ligated into the pGEX-KG BamHI-HindIII fragment and the prot ein of interest (p25(nck5a), which is highly susceptible to proteolytic deg radation when expressed and purified from bacteria) was cloned into the Bam HI site without a termination codon. The resulting plasmid construct, desig nated as pGST-p25(nck5a)-6His, with GST at the N-terminal and 6His at the C -terminal was expressed in Escherichia coli DH5 alpha and purified using a two-step procedure. We show that using Ni2+-NTA chromatography as a first p urification step and GSH-agarose chromatography as a second step, rather th an vice-versa, yields a highly purified intact protein that is free of any contaminating degraded protein product. The purified fusion protein is solu ble and fully active. (C) 2000 Academic Press.