We have designed and constructed a novel pair of bacterial co-expression vectors to facilitate the production of substantial amounts of recombinant multiprotein complexes for biochemical, biophysical, and structural studies. pOKD4 (kanamycin-resistant) and pOKD5 (ampicillin-resistant) are derivatives of pACYC177 cloning and pET26b expression vectors. As a result, pOKD4 and pOKD5 are T7-based expression plasmids containing the p15A origin of replication. This feature permits either pOKD4 or pOKD5 to co-exist in the same bacterial cell with most Escherichia coli expression vectors including the popular pET expression vectors. The pOKD4 and pOKD5 vectors have been engineered to possess exactly the same multiple cloning sites as pET26b thus allowing for the relatively easy shuttling of genes to and fro. The efficacy and versatility of this novel pair of co-expression vectors was successfully applied to the production of significant amounts of active DFF40/DFF45 heterodimeric protein complex in E. coli for detailed biochemical and structural studies. (C) 2004 Elsevier Inc. All rights reserved.