title: Production and secretion of Lactobacillus crispatus β-galactosidase in Pichia pastoris.
abstract: 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.
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; College of Life Sciences, Inner Mongolia Agricultural University, Huhhot 010018, China.
authors.forename: Chunming
authors.initials: C
authors.lastname: Nie
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Guofen
authors.initials: G
authors.lastname: Zhao
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Fan
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Ning
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001616:beta-Galactosidase
keywords: Extracellular secretion; Lactobacillus crispatus; Overlapping gene; Pichia pastoris; β-Galactosidase