Refolding, isolation and characterization of crystallizable human interferon-alpha 8 expression in Saccharomyces cerevisiae.
Human interferon-alpha 8 was expressed in Saccharomyces cerevisiae and found to accumulate intracellularly in an insoluble form. The protein could be solubilized and converted to a biologically active form with high yield by a denaturation-refolding procedure. The interferon-alpha 8 was further purified to apparent homogeneity by copper-chelate affinity chromatography and anion-exchange chromatography and fully characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, mass spectrometry, circular-dichroism (CD) spectroscopy and specific activity. Secondary-structure predictions from CD spectroscopy indicate that the molecule is correctly folded. Peptide mapping supported the correct sequence and the expected disulfide-bridge connectivity. The purified protein elutes on reversed-phase high-pressure liquid chromatography (RP-HPLC) as two peaks. Electrospray mass spectrometry and N-terminal sequence analysis of the minor component indicated the existence of an N-terminal acetyl group for the later eluting HPLC-component. In anti-viral assays, the two IFN forms were equally active. Hexagonal crystals of this interferon preparation could be obtained. On the basis of the electrophoretic mobility, HPLC profile, and biological activity assay, the crystalline material was judged to be identical to the uncrystallized interferon. Interferon in crystallized form was found to be stable for up to 24 months and, therefore, could be used for long-term storage, particularly for material intended for clinical use.
Di Marco
S
S
Department of Core Drug Discovery Technology, Ciba-Geigy, Ltd., Basle, Switzerland.
Fendrich
G
G
Meyhack
B
B
Grütter
M G
MG
D000595:Amino Acid Sequence; D001709:Biotechnology; D002942:Circular Dichroism; D003460:Crystallization; D004355:Drug Stability; D006801:Humans; D007370:Interferon Type I / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D007370:Interferon Type I; D011994:Recombinant Proteins