Expression, purification and characterization of human IFN-λ1 in Pichia pastoris

Interferon-lambda (IFN-λ) is a newly identified IFN family which belongs to the class II cytokines. The three members of this family represent antiviral activities like other IFNs. In the present study, recombinant human IFN-λ1 (rhIFN-λ1) was produced by using the methylotrophic yeast Pichia pastoris (P. pastoris) expression system. cDNAs encoding amino acids 23-200 or 20-200 of human IFN-λ1 were cloned and joined to sequence encoding the leader region (prepro segment) of the precursor of Saccharomyces cerevisiae α-factor. The two hybrid genes were subcloned into yeast integrative vector pAO815 separately to construct expression plasmids bearing four tandem copies of IFN-λ1 expression cassettes. The expression plasmids were then used to transform into P. pastoris strain GS115, resulting in recombinant strains GS115/IFNλ1P and GS115/IFNλ1G with Mut+ or Muts phenotype. rhIFN-λ1 was secreted into the medium upon methanol induction. In GS115/IFNλ1P, however, KEX2 cleavage for mature rhIFN-λ1 generation was inhibited by a proline at P′1 and the products were different from anticipation. GS115/IFNλ1G strain secreted two forms of mature rhIFN-λ1 with the same N-terminal sequence and different molecular weight. Periodic acid-Schiff (PAS) staining indicated that these proteins were glycosylated. The yield of low-glycosylated rhIFN-λ1 in GS115/IFNλ1G strain was approximately 65 mg l−1 in shaking flasks, representing around 57% of the total secreted proteins. rhIFN-λ1 was purified by cation exchange chromatography and gel filtration. The purified rhIFN-λ1 showed specific efficiency to activate signal transducer and activator of transcription 1 (STAT1) and STAT2 that was comparable to that of commercial IFNα2a.