A bifunctional vector suitable for both site-directed mutagenesis and recombinant expression of interferon-tau in Escherichia coli.
In order to produce workable quantities of a large number of mutant forms of recombinant ovine and bovine interferon-t (IFN-t), a bifunctional vector, pME-2, was developed, which combines a mutant selection system and a strong promoter providing controlled expression. An EcoRI/KpnI fragment containing the complete Trp promoter, a Shine-Dalgarno sequence, and an AT-rich region from the pTrp-2 expression vector was inserted into the large fragment of EcoRI/KpnI-digested pALTER-1 plasmid, which had been modified by eliminating a ClaI site. The pALTER-1 phagemid provides a highly efficient, antibiotic-dependent system for selection of mutant plaques. The existing T7 promoter was then eliminated from the recombinant phagemid to create the pME-2 vector. Ovine and bovine 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 designed to produce elongations, truncations, and single and multiple amino acid replacements in the protein products, mutant genes were selected in Escherichia coli BMH 71-18 and efficiently expressed in E. coli JM-101 in response to the inducer of the Trp promoter indole acetic acid. The recombinant IFN were solubilized from washed inclusion bodies in guanidinium-HCl and 2-mercaptoethanol and allowed to refold in aerated buffer. The procedure provides high yields of fully active, homogeneous IFN-t and can be accomplished within 1 week.
Department of Biochemistry, University of Missouri, Columbia 65211, USA.
J
J
Li
A P
AP
Alexenko
R M
RM
Roberts
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D007370:Interferon Type I / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011257:Pregnancy Proteins / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D012756:Sheep
D004274:DNA, Recombinant; D007370:Interferon Type I; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C020947:interferon tau