Expression of human interleukin-12 gene in Pichia pastoris

Human interleukin-12(hIL-12) is an immunoregulatory cytomine with multiple biological functions in human body, which consists of two disulfide-linked subunits, p40 and p35. Based on this structural character, yeast strain Pichia pastoris X33/p40 was constructed by transforming the expression vector pPICZ#alpha#C-p40 into Phichia pastoris X33 competent cells via LiCl transformation. Then the recombinant expression vector pPICZ#alpha#C-p35 was used to transform the Pichia pastoris X33/p40 with LiCl, the yeast strain Pichia pastoris X33/p40-p35 containing the integrated hIL-12 p40 and p35 cDNA was obtained by high concentration Zeocin selection (1000mg/L). After induction with 0.5% methanol, the hIL-12p40 and p35 subunits were expressed secretely and assembled as hIL-12p70 in the same cell. PHA-activated PBMC proliferation assay demonstrated that the hIL-12 expressed in Pichia pastoris had the bioactivity to induce the proliferation of PHA-activated PBMC. A new pathway to express a protein with two or more subunits through Pichia expression system was developed.