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Development of bicistronic expression system for the enhanced and reliable production of recombinant proteins in Leuconostoc citreum.
- Source :
-
Scientific reports [Sci Rep] 2018 Jun 11; Vol. 8 (1), pp. 8852. Date of Electronic Publication: 2018 Jun 11. - Publication Year :
- 2018
-
Abstract
- The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1 <superscript>st</superscript> cistron) followed by target genes (2 <superscript>nd</superscript> cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2 <superscript>nd</superscript> cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P <subscript>710V4</subscript> ) were successfully isolated. The usefulness of the engineered BCD with P <subscript>710V4</subscript> and eSD2 was further validated using three model proteins-glutathione-s-transferase, human growth hormone, and α-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.
- Subjects :
- Glutathione Transferase biosynthesis
Glutathione Transferase genetics
Green Fluorescent Proteins genetics
Human Growth Hormone biosynthesis
Human Growth Hormone genetics
Recombinant Proteins genetics
alpha-Amylases biosynthesis
alpha-Amylases genetics
Leuconostoc genetics
Recombinant Proteins biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 8
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 29891982
- Full Text :
- https://doi.org/10.1038/s41598-018-27091-z