Expression, purification and characterization of human IFN-lambda1 in Pichia pastoris.
Interferon-lambda (IFN-lambda) 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-lambda1 (rhIFN-lambda1) 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-lambda1 were cloned and joined to sequence encoding the leader region (prepro segment) of the precursor of Saccharomyces cerevisiae alpha-factor. The two hybrid genes were subcloned into yeast integrative vector pAO815 separately to construct expression plasmids bearing four tandem copies of IFN-lambda1 expression cassettes. The expression plasmids were then used to transform into P. pastoris strain GS115, resulting in recombinant strains GS115/IFNlambda1P and GS115/IFNlambda1G with Mut(+) or Mut(s) phenotype. rhIFN-lambda1 was secreted into the medium upon methanol induction. In GS115/IFNlambda1P, however, KEX2 cleavage for mature rhIFN-lambda1 generation was inhibited by a proline at [Formula: see text] and the products were different from anticipation. GS115/IFNlambda1G strain secreted two forms of mature rhIFN-lambda1 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-lambda1 in GS115/IFNlambda1G strain was approximately 65 mg l(-1) in shaking flasks, representing around 57% of the total secreted proteins. rhIFN-lambda1 was purified by cation exchange chromatography and gel filtration. The purified rhIFN-lambda1 showed specific efficiency to activate signal transducer and activator of transcription 1 (STAT1) and STAT2 that was comparable to that of commercial IFNalpha2a.
Xie
Yun Fei
YF
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao Street, Beijing, 100005, China.
Chen
Hong
H
Huang
Bing Ren
BR
D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D006367:HeLa Cells; D006801:Humans; D007372:Interferons; D007378:Interleukins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D000095022:Interferon Lambda
D016207:Cytokines; D017931:DNA Primers; D018076:DNA, Complementary; D007372:Interferons; D007378:Interleukins; D011993:Recombinant Fusion Proteins; C000624353:interferon-lambda, human; D000095022:Interferon Lambda