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- Le, T. N., et al.
(författare)
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Expression and simple purification strategy for the generation of antimicrobial active enterocin P from Enterococcus faecium expressed in Escherichia coli ER2566
- 2014
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Ingår i: Iranian Journal of Biotechnology. - : Armenian Green Publishing Co.. - 1728-3043 .- 2322-2921. ; 12:4, s. 16-25
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Tidskriftsartikel (refereegranskat)abstract
- Background: Enterocin-P of Enterococcus faecium P13 (EntP) is of great interest as a food preservative and medicine due to its non-toxicity and broad antimicrobial spectrum at various pH, as well as its excellent thermal stability. However, recombinant production of EntP is still in laboratory because of low productivity and complex purification process. Objectives: In this study, we aimed to develop efficient methods for high-level expression and convenient purification of the recombinant EntP.Materials and Methods: An artificially synthesized gene (entP) of 132 bp encoding mature enterocin P of E. faecium P13 was cloned in plasmid pTWIN1 under the control of an inducible T7lac promoter for expression of fusion protein EntP- Mxe GyrA mini-intein-chitin binding domain (CBD) (abbreviated by EntP-Int-CBD) in E. coli. Recombinant EntP was released from the fusion protein by DTT digestion and cleaned by distilled water and checked for anti-bacterial activity. Results: The fusion protein was highly expressed in insoluble form in E. coli at 37ºC with 0.05 mM IPTG induction. The insoluble fusion protein EntP-Int-CBD was easily prepared by cell sonication and centrifugation to remove soluble con- taminants. The repeat washing steps with Triton X-100 were applied to reduce contaminants. After DTT-induced self- digestion in urea 4 M, the EntP released from the fusion protein was insoluble in water and easier to be separated from sol- uble Int-CBD by centrifugation. The recombinant peptide was soluble in 20% 2-propanol in 0.1% trifluoroacetic acid (TFA) and exhibited strong anti- Listeria monocytogenes and Staphylococcus aureus activities.Conclusions: This study is the first report providing a simple, quick and straight forward procedure for heterologous production of functional and pure Enterocin P without using any chromatography columns in the purification process.
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| 4. |
- Wu, Ping-Hsun, et al.
(författare)
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The association between Single Nucleotide Polymorphisms of Klotho Gene and Mortality in Elderly Men: The MrOS Sweden Study
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- The Klotho (KL) gene is involved in phosphate homeostasis. Polymorphisms in this gene have been reported to be associated with the risk of cardiovascular disease. Here we used computational tools to predict the damage-associated single nucleotide polymorphisms (SNPs) in the human KL gene. We further investigated the association of SNPs in the KL gene and mortality in the Swedish multicenter prospective Osteoporotic Fractures in Men (MrOS) cohort. This study included 2921 men (aged 69-81 years) with mean 4.49 +/- 1.03 years follow-up. 18 SNPs in the KL gene were genotyped using Sequenom. These SNPs were identified by in silico tools for the coding and noncoding genome to predict the damaging SNPs. After quality analyses, SNPs were analyzed for mortality risk using two steps approach on logistic regression model screening and then Cox regression model confirmation. Two non-synonymous SNPs rs9536314 and rs9527025 were found to be potentially damaging SNPs that affect KL protein stability and expression. However, these two SNPs were not statistically significantly associated with all-cause mortality (crude Hazard ratio [HR] 1.72, 95% confidence interval [CI] 0.96-3.07 in rs9536314; crude HR 1.82, 95% CI 0.998-3.33 in rs9527025) or cardiovascular mortality (crude HR 1.52, 95% CI 0.56-4.14 in rs9536314; crude HR 1.54, 95% CI 0.55-4.33 in rs9527025) in additive model using Cox regression analysis. In conclusion, these two potentially damaging SNPs (rs9536314 and rs9527025) in the KL gene were not associated with all-cause mortality or cardiovascular mortality in MrOs cohort. Larger scales studies and meta-analysis are needed to confirm the correlation between polymorphisms of the KL gene and mortality.
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