Improvement of protein production in lactic acid bacteria using 5'-untranslated leader sequence of slpA from Lactobacillus acidophilus. Improvement in protein production using UTLS.
The 5'-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5' stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses alpha-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the alpha-amylase mRNA was proved by measuring alpha-amylase activity in the culture supernatant and the relative expression of alpha-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving alpha-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.
Narita
Junya
J
Division of Molecular Science, Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan.
Ishida
Saori
S
Okano
Kenji
K
Kimura
Sakurako
S
Fukuda
Hideki
H
Kondo
Akihiko
A
D020121:5' Untranslated Regions; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D019344:Lactic Acid / Q000378:metabolism*; D007779:Lactobacillus acidophilus / Q000378:metabolism*; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000516:alpha-Amylases / Q000378:metabolism
D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; C070683:surface layer protein A, Bacteria; D019344:Lactic Acid; D000516:alpha-Amylases