Open AccessHigh-level expression of an interferon alpha 2 gene cloned in phage M13mp7 and subsequent purification with a monoclonal antibody.
Author(s) -
Patrick M. Slocombe,
Andrew J. Easton,
Paul Boseley,
D. C. Burke
Publication year - 1982
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.79.18.5455
Subject(s) - microbiology and biotechnology , biology , alpha interferon , amino acid , lac operon , recombinant dna , escherichia coli , monoclonal antibody , nucleic acid sequence , peptide sequence , gene , biochemistry , antibody , interferon , virology , genetics
A cloned interferon alpha 2 (IFN-alpha 2) gene was partially digestd with Pvu II to give a fragment that was inserted into the HincII site of the lacZ gene of bacteriophage M13mp7. Two recombinant phages containing the IFN-alpha 2 sequences in the correct orientation for expression from the lac promoter were characterized in detail. DNA sequence analysis showed that the inserted IFN-alpha 2 gene was in phase with the initiation codon of the lacZ gene. The polypeptide product has an additional 19 amino amino acids at the amino terminus of the mature IFN-alpha 2. The first 11 amino acids originate from the amino terminus of beta-galactosidase, and the remaining 8 amino acids are part of the signal sequence of pre-IFN-alpha 2. Infection of Escherichia coli with these phage followed by induction of the lac promoter with isopropyl thiogalactoside gives high yields (up to 10(9) units/liter with an average of 1.5 X 10(8) units/liter) of the modified IFN-alpha 2. This was purified to homogeneity in a single step by immunochromatography using the monoclonal antibody NK2. The nonreduced product had an apparent molecular weight of 20,500 and was shown by immunoradiometric assay to have the same specific activity as IFN made in Namalwa cells. It exhibited the characteristic cross-species antiviral activity of IFN-alpha 2.