, ¦«1 Of Ifn-expression In Pichia Pastoris, Purification And Activity Determination And Its Receptor Signaling Pathway

IFN-λs are newly discovered interferon-like cytokines, including IFN-λ1(IL-299), IFN- λ2(IL-28A) and IFN- λ3(IL-28B). All of which possess intrinsic antiviral activity, and they will be reasonable candidates for new antiviral medicine, especially IFN- λ1. IFN- λ s are similar to members of the IL-10 gene family in genomic structure, but they are more closely related to type I IFNs at the amino acid level. IFN- λs display an evolutionary link between IL-10 family and type I IFNs. So a series of work was performed for IFN-λ1 gene cloning, expression, biological activity detection and the interaction between associated proteins.In this research, IFN-λ1 cDNA was cloned from peripheral blood lymphocytes by RT-PCR. The nucleotide sequence is the same as reported except for a base, and the amino acid sequence is fully identical to GenBank. IFN-λ1 was expressed in E.coli and Pichia pastoris systems in order to produce abundance of recombinant protein. But the expression level was very low in E. coli, which is probably due to the different preference of genetic codon usage in E.coli and human, or the toxicity of IFN- λ1 to E.coli. Then the expression plasmid pAO815-4 α F-IFN λ1 containing four copies of IFN λ1 was constructed and transformed into Pichia pastoris GS115. Recombinant human IFN λ1 (rhIFN λ1) was secreted into the medium at 28 mg/mL. In GS115, rhIFN λ1 was synthesized as a precursor of 263 amino acids containing an 85-residue leader peptide from Saccharomyces cerevisiae α -factor. Mature rhIFN λ1 with wild primary sequence should be produced if the precursor was cleaved properly by KEX2 protease. But the fusion protein was processed improperly, leading to three isoforms of rhIFN λ1 with different N-terminus. Two of which has 11aa and 9aa of leader peptide sequence protruding at the N-terminus respectively, and the third has a N-terminal 13aa-deletion. We speculated that it was the amino acid Pro after Glu-Lys-Arg inhibit KEX2 cleavage because X-Pro peptide bond possesses a high degree of resistance to any mammalian proteolytic enzyme. rhIFN λ1 was purified with a FPLC SP Sepharose Fast Flow column and the recovery was about 70%. The purified protein...