Synthesis of fusion and mature murine alpha interferons in Escherichia coli |
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Authors: | K A Kelley N B Raj M Kellum P M Pitha |
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Affiliation: | 1. Department of Microbiology, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria;2. Bioconversion and Renewable Energy Research Unit, University of Nigeria, Nsukka, Enugu State, Nigeria;1. Department of Geoscience and Remote Sensing, Delft University of Technology, 2628CN Delft, the Netherlands;2. State Key Laboratory of Information Engineering in Surveying, Mapping and Remote Sensing, Wuhan University, Wuhan 430079, China |
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Abstract: | Four murine interferon-alpha (MuIFN-alpha) genes (alpha 1, alpha 4, alpha 5, alpha 6) were previously identified and characterized. The coding regions of these IFN-alpha genes were inserted into bacterial expression vectors behind the lpp promoter under the control of the lac promoter-operator region, resulting in fusion peptides containing additional N-terminal amino acids (aa). Plasmids coding for the expression of mature IFN-alpha 1 and alpha 5 were also constructed using the same vector system, by inserting a 30-bp synthetic oligodeoxynucleotide, which contains a stop codon for the lpp gene, a ribosome-binding sequence and an ATG start codon for the IFN peptides. The amounts of IFN polypeptides synthesized in Escherichia coli were estimated in the maxi-cell system and their biological activities were measured on mouse and other mammalian cells. The yields of mature IFN produced in this vector were 2 to 4 X 10(6) units/liter; the antiviral activity of the majority of the MuIFNs on human and bovine cells was 100- to 1000-fold lower than on mouse cells. IFN-alpha 4, which contains an internal deletion of 5 aa, showed a lower antiviral activity than other MuIFNs on mouse cells. |
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