Construction And Expression Of The Gene Encoding Human Serum Album/Interleukin-11 Fusion Protein In Pichia Pastoris

Human interleukin-11(IL-11) is produced by PU-34 cells. It was initially described as a growth factor synergising with other factors in the regulation of hematopoiesis. The half-life of native IL-11 is short. In order to prolong the half-life, the fusion protein HSA-IL-11 was constructed by splicing the IL-11 to Human Serum Album (HSA) without peptide linker.The cDNA encoding IL-11 was obtained by RT-PCR with the template of total RNA purified from human fetal lung fibroblast, which was prepared from fresh tissue. These cells were stimulated with DMEM, then cloned into pMD19-Simple to construct the recombinant plasmid pMD19/IL-11.The fusion gene encoding HSA-IL-11 was amplified by overlap PCR extension using pMD19/IL-11 and pBlue/HSA (containing the gene encoding HSA) as the template, and then cloned into pBluescriptⅡKS(+) to construct the recombinant plasmid pBlue/HI. The pBlue/HI was digested with EcoRI and NotI, and ligated between the corresponding sites of pPIC9K, yielding the recombinant expressing plasmid pPHI11. Fidelity of the cloned DNA fragment was confirmed by DNA sequencing.The linearized plasmid pPHI11 digested by SalI was transformed into Pichia pastoris GS115 by electroporation. The transformants resisting 0.75 mg/mL of G418 were obtained. It was confirmed by PCR amplification that there was fusion gene encoding HSA-IL-11 in the transformant of P. pastoris GS115(pPHI11).The recombinant yeast of P. pastoris GS115(pPHI11) cultivated in BMGY medium and collected by centrifugation was resuspended in expression medium of BMMY, induced with methanol as the sole carbon source for 3 days at 30℃. SDS-PAGE analysis showed that the recombinant yeast expressed the protein with the similar molecular weight to the target fusion protein. Western-blot result was indicated that the expressed fusion protein HSA-IL-11 contained antigen both IL-11 and HSA. Quantitative analysis showed that the expression level of HSA-IL-11 was about 120 mg/L in expression suspension.The result of orthogonal test for rotation speed, temperature, pH, methanol concentration, and time showed the optimum expression condition for the recombinant yeast was as follows: inducing temperature of 30℃, methanol concentration of 5%; pH6. The expressing level was increased to 198 mg/L.The bioactivity of the expression supernatant was checked by B9-11/MTT method and the result showed that the fusion protein HSA-IL-11 can stimulate the division of B9-11 cells. The IL-11 activity was about 6.2×104 U/mg of the supernatant.