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Lactococcus lactis M4, a potential host for the expression of heterologous proteins.

Noreen N, Hooi WY, Baradaran A, Rosfarizan M, Sieo CC, Rosli MI, Yusoff K, Raha AR - Microb. Cell Fact. (2011)

Bottom Line: Plasmid profiling results indicated the lack of low molecular weight plasmids for strain M4.SDS-PAGE and Western blot analyses also confirmed the presence of GFP, demonstrating the potential of heterologous protein expression in M4.This strain has the potential to be developed into a new lactococcal host for the expression of heterologous proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.

ABSTRACT

Background: Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.

Results: Several bacterial strains were isolated from cow's milk and eight of those were identified as Lactococcus lactis by 16S rRNA sequence analysis. Antibiotic susceptibility tests that were carried out showed that 50% of the isolates had almost identical antibiotic resistance patterns compared to the control strains MG1363 and ATCC 11454. Plasmid profiling results indicated the lack of low molecular weight plasmids for strain M4. Competent L. lactis M4 and MG1363 were prepared and electrotransformed with several lactococcal plasmids such as pMG36e, pAR1411, pAJ01 and pMG36e-GFP. Plasmid isolation and RE analyses showed the presence of these plasmids in both M4 and the control strain after several generations, indicating the ability of M4 to maintain heterologous plasmids. SDS-PAGE and Western blot analyses also confirmed the presence of GFP, demonstrating the potential of heterologous protein expression in M4.

Conclusions: Based on the 16S rRNA gene molecular analysis, eight Gram-positive cocci milk isolates were identified as L. lactis subsp. lactis. One of the strains, L. lactis M4 was able to maintain transformed low molecular weight plasmid vectors and expressed the GFP gene. This strain has the potential to be developed into a new lactococcal host for the expression of heterologous proteins.

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Plasmid profile of lactococcal strains isolated from raw milk. Lane M: GeneRuler™ 1 kb DNA Ladder; Lane 1: M2; Lane 2: M4; Lane 3: M5; Lane 4: M6; Lane 5: M11; Lane 6: M12; Lane 7: M14; Lane 8: M16; Lane 9: MG1363; Lane 10: ATCC 11454. The size of plasmids from ATCC 11454 are depicted.
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Figure 3: Plasmid profile of lactococcal strains isolated from raw milk. Lane M: GeneRuler™ 1 kb DNA Ladder; Lane 1: M2; Lane 2: M4; Lane 3: M5; Lane 4: M6; Lane 5: M11; Lane 6: M12; Lane 7: M14; Lane 8: M16; Lane 9: MG1363; Lane 10: ATCC 11454. The size of plasmids from ATCC 11454 are depicted.

Mentions: Two of the eight lactococcal strains studied gave plasmid profiles detectable with the method used (Figure 3). These (M12 and M14) lactococcal strains contained several plasmids with various sizes, ranging from 1.6 kb to ~46 kb, and gave different banding patterns that can differentiate them. However, the plasmid extraction of the other six strains was unsuccessful although repeated several times. The plasmid-free strain MG1363 and the nisin-producing strains ATCC 11454 were used as control for the plasmid extraction. The ATCC strain was reported to carry plasmids with the size of 48.2, 44.1, 33.2, 29.4, 5.5 and 2.3 kb [25].


Lactococcus lactis M4, a potential host for the expression of heterologous proteins.

Noreen N, Hooi WY, Baradaran A, Rosfarizan M, Sieo CC, Rosli MI, Yusoff K, Raha AR - Microb. Cell Fact. (2011)

Plasmid profile of lactococcal strains isolated from raw milk. Lane M: GeneRuler™ 1 kb DNA Ladder; Lane 1: M2; Lane 2: M4; Lane 3: M5; Lane 4: M6; Lane 5: M11; Lane 6: M12; Lane 7: M14; Lane 8: M16; Lane 9: MG1363; Lane 10: ATCC 11454. The size of plasmids from ATCC 11454 are depicted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101652&req=5

Figure 3: Plasmid profile of lactococcal strains isolated from raw milk. Lane M: GeneRuler™ 1 kb DNA Ladder; Lane 1: M2; Lane 2: M4; Lane 3: M5; Lane 4: M6; Lane 5: M11; Lane 6: M12; Lane 7: M14; Lane 8: M16; Lane 9: MG1363; Lane 10: ATCC 11454. The size of plasmids from ATCC 11454 are depicted.
Mentions: Two of the eight lactococcal strains studied gave plasmid profiles detectable with the method used (Figure 3). These (M12 and M14) lactococcal strains contained several plasmids with various sizes, ranging from 1.6 kb to ~46 kb, and gave different banding patterns that can differentiate them. However, the plasmid extraction of the other six strains was unsuccessful although repeated several times. The plasmid-free strain MG1363 and the nisin-producing strains ATCC 11454 were used as control for the plasmid extraction. The ATCC strain was reported to carry plasmids with the size of 48.2, 44.1, 33.2, 29.4, 5.5 and 2.3 kb [25].

Bottom Line: Plasmid profiling results indicated the lack of low molecular weight plasmids for strain M4.SDS-PAGE and Western blot analyses also confirmed the presence of GFP, demonstrating the potential of heterologous protein expression in M4.This strain has the potential to be developed into a new lactococcal host for the expression of heterologous proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.

ABSTRACT

Background: Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.

Results: Several bacterial strains were isolated from cow's milk and eight of those were identified as Lactococcus lactis by 16S rRNA sequence analysis. Antibiotic susceptibility tests that were carried out showed that 50% of the isolates had almost identical antibiotic resistance patterns compared to the control strains MG1363 and ATCC 11454. Plasmid profiling results indicated the lack of low molecular weight plasmids for strain M4. Competent L. lactis M4 and MG1363 were prepared and electrotransformed with several lactococcal plasmids such as pMG36e, pAR1411, pAJ01 and pMG36e-GFP. Plasmid isolation and RE analyses showed the presence of these plasmids in both M4 and the control strain after several generations, indicating the ability of M4 to maintain heterologous plasmids. SDS-PAGE and Western blot analyses also confirmed the presence of GFP, demonstrating the potential of heterologous protein expression in M4.

Conclusions: Based on the 16S rRNA gene molecular analysis, eight Gram-positive cocci milk isolates were identified as L. lactis subsp. lactis. One of the strains, L. lactis M4 was able to maintain transformed low molecular weight plasmid vectors and expressed the GFP gene. This strain has the potential to be developed into a new lactococcal host for the expression of heterologous proteins.

Show MeSH
Related in: MedlinePlus