Production and secretion of Lactobacillus crispatus beta-galactosidase in Pichia pastoris

文献类型： 外文期刊

第一作者： Nie, Chunming

作者： Nie, Chunming;Liu, Bo;Zhang, Yuhong;Fan, Xiaohu;Ning, Xiaoyan;Zhang, Wei;Nie, Chunming;Zhao, Guofen;Fan, Xiaohu

作者机构：

关键词： Lactobacillus crispatus;beta-Galactosidase;Extracellular secretion;Overlapping gene;Pichia pastoris

期刊名称：PROTEIN EXPRESSION AND PURIFICATION （ 影响因子：1.65； 五年影响因子：1.548 ）

ISSN： 1046-5928

年卷期： 2013 年 92 卷 1 期

页码：

收录情况： SCI

摘要： Lactobacillus beta-galactosidases are mostly heterodimeric proteins, which are encoded by the two overlapping genes, lacL and lacM, and produced in recombinant prokaryotic systems for higher yield. This is the first report on the expression of a heterodimeric beta-galactosidase from Lactobacillus crispatus B470 in Pichia pastoris. The overlapping consecutive genes, lacL and lacM, that shared 17 nucleotides were cloned from the genomic DNA of L. crispatus. A recombinant plasmid harboring both expression cassettes of lacL and lacM was constructed and transformed into P. pastoris GS115 competent cells. Two recombinant P. pastoris strains (GSLac01 and GSLac02) showed the highest beta-galactosidase activities of 24.5 and 31.0 U/ml in the culture supernatants, respectively. The recombinant beta-galactosidase (LcLacLM) from GSLac02 was purified to electrphoretic homogeneity by ion-exchange chromatography and molecular sieve chromatography. Similar to most Lactobacillus beta-galactosidases that operate at moderately thermophilic and weak acid to neutral conditions, LcLacLM showed optimal activity at 50 degrees C and pH 5.5-6.5. It's the first report on functional and secretory expression of LacLM-type beta-galactosidase in eukaryotic system. This strategy might be applied to the expression of other overlapping genes. (C) 2013 Elsevier Inc. All rights reserved.

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