Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica.
Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica.
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DOI：
10.1002/bit.10464
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发表时间：
2003-02
影响因子：
3.8
通讯作者：
Brian E Mayson;D. Kilburn;B. Zamost;C. Raymond;G. Lesnicki
中科院分区：
文献类型：
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作者：
Brian E Mayson;D. Kilburn;B. Zamost;C. Raymond;G. Lesnicki
甲基营养酵母甲醇毕赤酵母（Pichia methanolica）可以在甲醇诱导型酒精氧化酶（AUG1）启动子的调控下高水平表达重组基因。诱导阶段的甲醇浓度直接影响细胞生长和蛋白质产量。在葡萄糖/甲醇混合补料分批培养表达人转铁蛋白n-叶蛋白的甲醇假单胞菌中，研究了在线监测和控制系统控制的不同甲醇浓度。利用的PMAD18甲醇单胞菌菌株是染色体AUG1基因位点的敲除，导致甲醇利用缓慢的表型。在1.0% （v/v）甲醇浓度下，每升培养物中最大生长100 g干细胞重量。在0.7% （v/v）甲醇浓度的培养基中，重组基因的表达量最大，发酵72小时后，每升转铁蛋白的最大体积产量为450 mg。
The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.