[Beta-galactosidase gene from Lactobacillus delbrueckii subsp. bulgaricus gets non-fusion expression in Escherichia coli].
OBJECTIVE
To construct the food-grade recombinant probiotic strain with high activity beta-galactosidase, the beta-galactosidase gene (lacZ)from Lactobacillus delbrueckii subsp. bulgaricus was in non-fusion expressed in Escherichia coli.


METHODS
From Lb. delbrueckii subsp. bulgaricus lacZ gene, the DNA sequence containing Shine-Dalgarno (SD) and ATGA sequences between upstream 18 bp and downstream 1 bp at start codon ATG was selected as upstream primer for PCR amplifying lacZ gene. Then lacZ cDNA was inserted into expression plasmid pMG36e to construct recombinant expression plasmid. Recombinant plasmids were introduced into E. coli, and positive clones were screened. To identify the gene recombination, the recombinant plasmid was cut by restriction enzyme and sequenced. To identify the protein expression, the beta-galactosidase activities of recombinant strains were determined.


RESULTS
The restriction maps of recombinant plasmids were acceptable. The gene inserted into the recombinant plasmid had more than 99% homology with the lacZ gene of Lb. delbrueckii subsp. bulgaricus. The enzyme activity and enzyme activity ratio of E. coli DH5 alpha carrying pMG36e-lacZ 1.1480 were 3.074 U/mL and 6.939 U/mg pro respectively. The enzyme activity and enzyme activity ratio of E. coli DH5a carrying pMG36e-lacZ wch9901 were 4.755 U/mL and 8.537 U/mg pro respectively.


CONCLUSIONS
lacZ from Lb. delbrueckii subsp. bulgaricus have gotten non-fusion expression in E. coli. The SD and ATGA sequences we selected can introduce lacZ non-fusion expression in E. coli.
Wang
Chuan
C
Department of Medical Technology, West China School of Public Health, Sichuan University, Chengdu 610041, China.
Zhang
Chao-wu
CW
Yu
Qian
Q
Liu
Heng-chuan
HC
Pei
Xiao-fang
XF
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis*; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D001616:beta-Galactosidase