A BIFUNCTIONAL VECTOR SUITABLE FOR BOTH SITE-DIRECTED MUTAGENESIS ANDRECOMBINANT EXPRESSION OF INTERFERON-TAU IN ESCHERICHIA-COLI

In order to produce workable quantities of a large number of mutant fo rms of recombinant ovine and bovine interferon-t (IFN-t), a bifunction al vector, pME-2, was developed, which combines a mutant selection sys tem and a strong promoter providing controlled expression. An EcoRI/Kp nI fragment containing the complete Trp promoter, a Shine-Dalgarno seq uence, and an AT-rich region from the pTrp-2 expression vector was ins erted into the large fragment of EcoRI/KpnI-digested pALTER-1 plasmid, which had been modified by eliminating a ClaI site. The pALTER-1 phag emid provides a highly efficient, antibiotic-dependent system for sele ction of mutant plaques. The existing T7 promoter was then eliminated from the recombinant phagemid to create the pME-2 vector. Ovine and bo vine IFN-t genes lacking the coding region for the signal peptide, but with an ATG codon ahead of the open reading frame, were inserted into the multicloning site of pME-2. Following site-directed mutagenesis d esigned to produce elongations, truncations, and single and multiple a mino acid replacements in the protein products, mutant genes were sele cted in Escherichia coli BMH 71-18 and efficiently expressed in E. col i JM-101 in response to the inducer of the Trp promoter indole acetic acid. The recombinant IFN were solubilized from washed inclusion bodie s in guanidinium-HCl and 2-mercaptoethanol and allowed to refold in ae rated buffer. The procedure provides high yields of fully active, homo geneous IFN-t and can be accomplished within 1 week. (C) 1995 Academic Press, Inc.