摘要：Lactoferrin (LF) is a multifunctional iron-binding glycoprotein which is found in high concentrations in milk. Recombinant human LF (rhLF) may provide health benefits. In the present study, we report an optimization of the production system of recombinant human lactoferrin that has enabled the production of recombinant protein at levels up to 1200 mg$\cdot$L$^{-1}$. The hLF was expressed in the methylotrophic yeast Pichia pastoris using the pPIC9K vector. The expression level of recombinant hLF (rhLF) was improved significantly by mixed methanol/glycerol feeding at the ratio of 4:1 during the induction phase of high cell-density fermentation. A yield of approximately 1200 mg$\cdot$L$^{-1}$ was obtained in fed-batch fermentation with this system, which was much higher than the reported values for other systems (1 mg$\cdot$L$^{-1}$ to 115 mg$\cdot$L$^{-1}$). The rhLF was purified via ion-exchange chromatography using SP Sepharose$^{\rm TM}$ Fast Flow and has a similar molecular mass of 80 kg$\cdot$mol$^{-1}$ to native hLF. The level of glycosylation of the recombinant protein is similar to that of the native protein.
