Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum
| Autor: | Ling Li, Ki Jun Jeong, Nam Soo Han, Yeon Jeong Son, Ae Jin Ryu |
|---|---|
| Jazyk: | angličtina |
| Předmět: |
0301 basic medicine
clone (Java method) Sequence analysis 030106 microbiology FACS Green Fluorescent Proteins Mutagenesis (molecular biology technique) Plasmid engineering Bioengineering Biology medicine.disease_cause Polymerase Chain Reaction Applied Microbiology and Biotechnology Lactic acid bacteria Leuconostoc citreum High copy plasmid Plasmidengineering 03 medical and health sciences Plasmid Shuttle vector Bacterial Proteins medicine Leuconostoc Escherichia coli Research biology.organism_classification Molecular biology Recombinant Proteins Plasmids Biotechnology |
| Zdroj: | Microbial Cell Factories MICROBIAL CELL FACTORIES(15) |
| ISSN: | 1475-2859 |
| DOI: | 10.1186/s12934-015-0400-8 |
| Popis: | Background: Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc. Results: Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C -> T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and alpha-amylase. Conclusions: The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries. |
| Databáze: | OpenAIRE |
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