重组牛乳铁蛋白功能片段在毕赤酵母中的表达及高密度发酵

Recombinant bovine lactoferrin functional fragment expressed in Pichia pastoris and high-cell-density fermentation

该研究合成了密码子优化后的牛乳铁蛋白功能片段(bovine lactoferrin functional fragment, BlfFf ),转入毕赤酵母GS115中重组表达并筛选抗性菌株,通过镍亲和层析纯化BlfFf,以Western Blot、液质联用和ELISA对重组蛋白进行鉴定及检测。比较了5 L发酵罐生产中3种不同高密度发酵培养基对BlfFf生产的影响。结果表明:重组转化子正确表达了BlfFf。镍亲和层析中,150 mmol/L咪唑洗脱可得到电泳纯37 kDa的目标条带。在3种高密度发酵培养基中,RDM最优,诱导48 h,菌体湿细胞密度达到297 g/L,BlfFf表达量为38.1 mg/L。

This study aimed to express bovine lactoferrin functional fragments (BlfFf) in Pichia pastoris and investigate high-cell-density fermentation. The codon-optimized BlfFf was synthesized and transferred into P. pastoris GS115 for recombinant expression,followed by resistant strain screening. The BlfFf was purified with Ni affinity chromatography,and identified and determined by Western Blot,LC-MS,and ELISA assays. The effects of three different high-cell-density fermentation media on BlfFf production in a 5 L fermentor were compared. The results showed that the engineered yeast expressed BlfFf correctly,and a 37 kDa electrophoretic pure target band was obtained by elution with 150 mmol/L imidazole. Moreover,RDM was found to be the optimal culture medium. After 48 h induction,the wet cell density reached 297 g/L and the yield reached 38.1 mg/L. In conclusion,the recombinant P. pastoris was successfully constructed to produce BlfFf in RDM medium for high-cell-density fermentation,which shows its industrial application potentials.