Nie Chunming, Liu Bo, Zhang Yuhong, Zhao Guofen, Fan Xiaohu, Ning Xiaoyan, Zhang Wei
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; College of Life Sciences, Inner Mongolia Agricultural University, Huhhot 010018, China.
Protein Expr Purif. 2013 Nov;92(1):88-93. doi: 10.1016/j.pep.2013.08.019. Epub 2013 Sep 6.
Lactobacillus β-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 β-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 β-galactosidase activities of 24.5 and 31.0 U/ml in the culture supernatants, respectively. The recombinant β-galactosidase (LcLacLM) from GSLac02 was purified to electrphoretic homogeneity by ion-exchange chromatography and molecular sieve chromatography. Similar to most Lactobacillus β-galactosidases that operate at moderately thermophilic and weak acid to neutral conditions, LcLacLM showed optimal activity at 50°C and pH 5.5-6.5. It's the first report on functional and secretory expression of LacLM-type β-galactosidase in eukaryotic system. This strategy might be applied to the expression of other overlapping genes.
乳酸杆菌β-半乳糖苷酶大多是异源二聚体蛋白，由两个重叠基因lacL和lacM编码，并在重组原核系统中生产以获得更高产量。这是关于卷曲乳杆菌B470的异源二聚体β-半乳糖苷酶在毕赤酵母中表达的首次报道。从卷曲乳杆菌的基因组DNA中克隆出共享17个核苷酸的重叠连续基因lacL和lacM。构建了一个携带lacL和lacM两个表达盒的重组质粒，并将其转化到毕赤酵母GS115感受态细胞中。两个重组毕赤酵母菌株（GSLac01和GSLac02）在培养上清液中的β-半乳糖苷酶活性最高，分别为24.5和31.0 U/ml。通过离子交换色谱和分子筛色谱将来自GSLac02的重组β-半乳糖苷酶（LcLacLM）纯化至电泳纯。与大多数在中等嗜热和弱酸至中性条件下起作用的乳酸杆菌β-半乳糖苷酶相似，LcLacLM在50°C和pH 5.5 - 6.5下表现出最佳活性。这是关于LacLM型β-半乳糖苷酶在真核系统中功能和分泌表达的首次报道。该策略可能适用于其他重叠基因的表达。
