Cloning and Heterologous Expression of Extracellular Plantaricin F Produced by Lactobacillus plantarum S34 Isolated from “Bekasam” in Lactococcus lactis

Mustopa, Apon Zaenal; Murtiyaningsih, Hidayah; Fatimah, Fatimah; Suharsono, Suharsono

doi:10.5454/mi.10.3.3

Cited by 6 publications

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“…Furthermore, 10 ng/ml nisin was applied to optimize the heterologous expression level. Previous studies reported the construction of bacteriocins from various origins (plantaricin E, F, and W), which were successfully expressed in L. lactis, generating mature form at transcription and translational level [19,41,42]. Another studies also reported that nisin concentration at 10 ng/ml enhanced protein expression level 4.59x of native HBcAg gene [20], and conversely codon-optimized HBcAg gene was reported to produce the highest expression level with 50 ng/ml nisin concentration [17].…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression of Interferon α-2b in Lactococcus lactis and its Biological Activity against Colorectal Cancer Cells

Meilina¹,

Budiarti²,

Mustopa³

et al. 2021

Kor. J. Microbiol. Biotechnol

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Type I Interferons (IFNα) are known for their role as biological anticancer agents owing to their cell-apoptosis inducing properties. Development of an appropriate, cost-effective host expression system is crucial for meeting the increasing demand for proteins. Therefore, this study aims to develop codon-optimized IFNα-2b in L. lactis NZ3900. These cells express extracellular protein using the NICE system and Usp 45 signal peptide. To validate the mature form of the expressed protein, the recombinant IFNα-2b was screened in a human colorectal cancer cell line using the cytotoxicity assay. The IFNα-2b was successfully cloned into the pNZ8148 vector, thereby generating recombinant L. lactis pNZ8148-SP Usp45 -IFNα-2b. The computational analysis of codon-optimized IFNα-2b revealed no mutation and amino acid changes; additionally, the codon-optimized IFNα-2b showed 100% similarity with native human IFNα-2b, in the BLAST analysis. The partial size exclusion chromatography (SEC) of extracellular protein yielded a 19 kDa protein, which was further confirmed by its positive binding to anti-IFNα-2b in the western blot analysis. The crude protein and SEC-purified partial fraction showed IC 50 values of 33.22 μg/ml and 127.2 μg/ml, respectively, which indicated better activity than the metabolites of L. lactis NZ3900 (231.8 μg/ml). These values were also comparable with those of the regular anticancer drug tamoxifen (105.5 μg/ml). These results demonstrated L. lactis as a promising host system that functions by utilizing the pNZ8148 NICE system. Meanwhile, codonoptimized usage of the inserted gene increased the optimal protein expression levels, which could be beneficial for its large-scale production. Taken together, the recombinant L. lactis IFNα-2b is a potential alternative treatment for colorectal cancer. Furthermore, its activity was analyzed in the WiDr cell line, to assess its colorectal anticancer activities in vivo.

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Section: Discussionmentioning

confidence: 99%

Heterologous Expression of Interferon α-2b in Lactococcus lactis and its Biological Activity against Colorectal Cancer Cells

Meilina¹,

Budiarti²,

Mustopa³

et al. 2021

Kor. J. Microbiol. Biotechnol

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“…In accordance to the results of Lages et al, 23 the administration of 10 ng/ml nisin induction in L. lactis increased gene expression, resulting in more protein than the non-induced. The concentrations of nisin has compared in the range between 0.5 and 5 ng/ml to induce the expression genes of antibacterial in L. lactis, and obtained the result that the highest of antibacterial was produced by L. lactis induced with nisin at 5 ng/ml by Mustopa et al 24 Moreover, codon optimization can also be performed. Nisin concentration required for induction is very low, between 0.5-10 ng/ml, while the high concentration of nisin of more than 10 ng/ml used will have negative effect on the host because it can suppress the growth of L. lactis.…”

Section: Discussionmentioning

confidence: 99%

The production of SPusp45-MSP-1<sub>19</sub> gene construct and its recombinant protein in <em>Lactococcus lactis</em> to be used as a malaria vaccine

et al. 2018

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Background: Merozoite surface protein 1 (MSP-1) is a major protein used by the Plasmodium during red blood cells invasion in malaria. MSP-119, one of MSP-1 is highly conserved, and it is a potential malaria vaccine candidate because the monoclonal antibodies are capable blocking erythrocyte invasion in vitro. The aim of this study was to produce MSP-119 gene construct and the recombinant protein in Lactococcus lactis.Methods: Usp45-MSP-119, derived from codon optimization and the synthetic gene, was inserted into the pMAT cloning vector. A vector expressing MSP-119 included usp45 has been constructed by the manipulation of recombinant DNA using restriction enzymes. The MSP-119 protein was expressed to 45% ammonium sulfate precipitation and purified using Sephadex-G50 gel filtration chromatography. The expressed protein was characterized by SDS-PAGE and dot blot.Results: usp45-MSP-119 gene was amplified using specific primers and inserted into the multiple cloning sites in the expression vector pNZ8148 with size 3,538 bp as a recombinant vector. The protein of MSP-119 was successfully expressed in L. lactis with molecular weight of 10.45 kDa. The dot blot was tested in 3 different comparisons between the host cells, non-induced cells, and induced cells with 10 ng/ml nisin. The results showed that 10 ng/ml nisin gave a positive reaction as detected by dot blot assay.Conclusion: This study confirmed that the usp45-MSP-119 gene was successfully inserted into the multiple cloning sites of the pNZ8148 expression vector and the MSP-119 protein expressed in the NICE system of the L. lactis host cell.

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“…The new L. plantarum EK11 strain analyzed in our study exhibits genetic predisposition for the production of three important bacteriocins (PlnG, PlnEF, and PlnA). The biosynthesis of plantaricin J (PlnJ) and plantaricin K is induced by the activity of plantaricin A (PlnA), which determines the proper functioning of the ABC transporter responsible for plantaricins transport outside the cell (Anderssen et al, 1998; Mustopa et al, 2016). This indicates a direct association between the number of genes involved in the biosynthesis of plantaricins in a bacterial strain and its bactericidal or bacteriostatic potential against specific pathogens (Rizello et al, 2014;).…”

Section: Detection Of Genes Encoding Selected Bacteriocinsmentioning

confidence: 99%

Possibility of Reinforcement the Functional Potential of Vegetable Juices with the use of Novel Strain Lactiplantibacillus Plantarum EK11 Isolated from an Unconventional Fermented Food Matrix

Skrzypczak

Gustaw

Schwonke

et al. 2021

Acta Universitatis Cibiniensis. Series E: Food Technology

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The study investigated the suitability of a novel strain Lactiplantibacillus plantarum EK11 for obtaining fermented tomato and beetroot juices with improved functional potential. EK11 had the capability of dynamic acidification of pasteurized vegetable beverages. The lowest values of pH were noted in juices after 48 h of fermentation with the probiotic L. plantarum 299v (pH=3.72±0.01 in beet juice and pH=3.43±.0.01 in tomato juice). The fermentation increased the lycopene content in tomato juices from 27.90±0.31µg mL−1 (after 24-h fermentation by strain EK11) to 116.86 ±0.19 µg mL−1 (final products obtained using strain 299v after 7-day cold storage). The process contributed to changes in the betanin and vulgaxanthin-I concentration in beetroot beverages. All fermented products exhibited antioxidative activity, i.e. 50% inhibition of 1,1-diphenyl-2-picrylhydrazyl free radicals. Moreover, three genes involved in the biosynthesis of bacteriocins were detected in the novel strain EK11, which exhibits functional and technological potential for the production of fermented foods.

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Cloning and Heterologous Expression of Extracellular Plantaricin F Produced by Lactobacillus plantarum S34 Isolated from “Bekasam” in Lactococcus lactis

Abstract: Submission ID: 1079439462File name: logous_Expression_of _Extracellular_Plantaricin_F_in_L.lactis.pdf (2.14M) Word count: 6759Character count: 35493

Cited by 6 publications

References 0 publications

Heterologous Expression of Interferon α-2b in Lactococcus lactis and its Biological Activity against Colorectal Cancer Cells

Heterologous Expression of Interferon α-2b in Lactococcus lactis and its Biological Activity against Colorectal Cancer Cells

The production of SPusp45-MSP-1<sub>19</sub> gene construct and its recombinant protein in <em>Lactococcus lactis</em> to be used as a malaria vaccine

Possibility of Reinforcement the Functional Potential of Vegetable Juices with the use of Novel Strain Lactiplantibacillus Plantarum EK11 Isolated from an Unconventional Fermented Food Matrix

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