Expression Of Recombinant Human Interleukin-29 Mutants In Pichia Pastoris And Their Preliminary Antitumor Activity Analysis

IL-29(Interleukin-29) activates JAK-STAT signaling pathway which is shared with type I IFNs after binding with its specific receptor complex. For this reason, IL-29 presents some similar common biological activities with type I IFNs, such as immunoregulatory, antiviral and antitumor activity. Meanwhile, another study finds that the distribution of target cells of IL-29 in the body are restricted. All these findings suggest that IL-29 might be a potentially powerful antitumor drug for cancer therapy with little side-effects compared to type I IFNs.Compared with IL-28 B, four amino acid residues of IL-29 were detected at the positions in helices A and F: Lys-33, Arg-35, Lys-43 and Ala-143. Interestingly, mutagenesis at these positions of IL-28 B revealed a profound impact on its antitumor activity. Accordingly, it has been hypothesized that point mutations at positions Lys-33 and Arg-35 in IL-29 could influence its antitumor activity. In this work, three h IL-29 mutants were created at positions Lys-33 and Arg-35 by site-directed mutagenesis based on the corresponding residues of IL-28 B.The genes encoding the mature peptide of h IL-29 mutants were constructed by megaprimer PCR, inserted into p PIC9 KM and then transformed into Pichia pastoris for expression. The high-producing strains were obtained and verified by SDS-PAGE and Western blotting.After the verification of GS115/p PIC9KM-h IL-29 mut, the optimization of fermentation conditions of rh IL-29 mut were preliminary researched. According to the orthogonal results, we found that the optimal fermentation conditions were 1.5% methanol of p H value 7 at 26 ℃ for 60 h.After the purification, the recombinant human interleukin-29 mutant proteins(K33R, R35 K and K33R/R35K) were obtained. HPLC analyses showed that their purity were 98.59%,97.62% and 98.12%, respectively. The preliminary antitumor activity analyses exhibited that the K33R/R35K(1000 ng/m L) can inhibit the significant tumor cell proliferation,with the inhibition rate of 40.09±2.81% of BEL-7401, 38.11±2.67% of HCT-8 and 29.31±2.14% of SGC-7901, separately.