Production, purification and characterization of recombinant human interferon γ

Abstract

An essentially three-step chromatographic purification procedure, i.e., ion-exchange, immobilized metal ion affinity and size-exclusion chromatography, is described for the purification to homogeneity of recombinant human interferon-γ (rhIFN-γ) from the inclusion bodies produced in genetically transformed Escherichia coli cells. Batchwise adsorption of the cloudy solution of renatured rhIFN-γ obviated the need for high-speed centrifugation to clarify the suspension. This step effectively removed about 70% of extraneous protein impurities. The established purification process is reproducible and leads to a total recovery of 32%. Pilot-scale processing of E. coli cells grown in a 30-1 fermentor gave about 70 mg of a homogeneous preparation of rhIFN-γ. The specific biological activity of purified rhIFN-γ is ca. 3.4 · 10 7 I.U./mg protein, which is comparable to that of its natural counterpart. It is basic protein (p I > pH 9) with a monomer relative molecular mass of 15 000. It behaves, however, as a dimer on size-exclusion chromatography. Its partial NH 2-terminal sequence is identical with that established for the rhIFN-γ. However, its amino acid composition and its relative molecular mass (15 067 as determined by electrospray mass spectrometry) indicate that the purified protein is a truncated form lacking fifteen amino acid residues from its carboxyl-terminal side. This modification does not seem to have any adverse effect on its biological potency. The levels of DNA, bacterial endotoxins and Ni(II) ions in the final product were determined.