Engineered disulfide bond greatly increases specific activity of recombinant murine interferon-beta.
Unlike other species of interferon-beta (IFN-beta) mouse (Mu) IFN-beta has no naturally occurring intramolecular disulfide bond. When expressed in Escherichia coli, MuIFN-beta appears to exhibit instability and low activity. To increase its activity, we engineered a pair of cysteines into recombinant MuIFN-beta to test whether this change would improve its antiviral activity. In the absence of detailed structural data, the optimal placement of cysteines was determined by sequence comparison with other species of IFN-beta. While MuIFN-beta has only a single cysteine (at position 17), other species of IFN-beta have three cysteines, at positions 17, 31, and 141 (numbering based on consensus sequence), a disulfide bond being formed between the latter two residues. Thus, we introduced cysteines into MuIFN-beta at positions 29 and 136, which correspond to positions 31 and 141 of the consensus sequence. When the variant form of MuIFN-beta was expressed in bacteria and purified, we found that the additional cysteines greatly increased the antiviral activity. Further improvement was obtained by replacement of the Cys-17 with a serine. In this manner a specific activity approximately 15-fold that of the wild-type recombinant molecule was achieved.
Day
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C
National Institutes of Health, Institute of Allergies & Infectious Diseases, Rockville, MD 20852.
Schwartz
B
B
Li
B L
BL
Pestka
S
S
D000818:Animals; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D016899:Interferon-beta / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010948:Viral Plaque Assay
D004220:Disulfides; D011994:Recombinant Proteins; D016899:Interferon-beta