The Expression of Codon Optimised Hepatitis B Core Antigen (HBcAg) of Subgenotype B3 Open Reading Frame in Lactococcus lactis

Mustopa, Apon Zaenal; Wijaya, Sri Kartika; Ningrum, Ratih Asmana; Agustiyanti, Dian Fitria; Triratna, Lita; Alfisyahrin, Wida Nurul

doi:10.4014/mbl.1806.06004

Cited by 2 publications

(4 citation statements)

References 18 publications

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“…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%

“…2). The increasing GC content of codon-optimized gene also conveniently expressed HBcAg gene in L. lactis, with the total protein raises of 10−20% compared to the native gene [17]. Moreover, the expressed target protein was one of the crucial factors in obtaining the expected yields.…”

Section: Discussionmentioning

confidence: 99%

“…The elution protein samples were fractionated by 100 mM Tris-HCl (pH 8) with a 1 ml/min flow-rate. Protein fractionation were collected and analyzed with spectrophotometer at 280 nm [17].…”

Section: Expression and Purification Of Recombinant Ifnα-2b Proteinmentioning

confidence: 99%

“…Currently, there is still a limited report on recombinant IFNα-2b with codon-optimized in the food-grade host system. Previous studies have been focusing on codon-optimized HBcAg synthetic gene in food-grade host system, and resulting in 1.5−3.2 folds of protein expression level [17]. This study reports the codon-optimized IFNα-2b in L. lactis as a component of live therapeutic protein.…”

Section: Introductionmentioning

confidence: 99%

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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%

Section: Discussionmentioning

confidence: 99%

“…The elution protein samples were fractionated by 100 mM Tris-HCl (pH 8) with a 1 ml/min flow-rate. Protein fractionation were collected and analyzed with spectrophotometer at 280 nm [17].…”

Section: Expression and Purification Of Recombinant Ifnα-2b Proteinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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Genetic construction of HBsAg gene subgenotype B3 in Lactococcus lactis as hepatitis B vaccine candidate

Mustopa

Putri²,

Kusdianawati³

et al. 2021

IOP Conf. Ser.: Earth Environ. Sci.

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Hepatitis B is an inflammatory liver disease caused by HBV (Hepatitis B Virus). Hepatitis B surface antigen (HBsAg) induces immune system forming antibodies. HBV subgenotype B3 is common in Asian Countries. Thus, the development of HBsAg subgenotype B3 vaccine was done because its prevalence is high in Indonesia (especially in Javanese) and other Asian countries. The research methods were preparation of the HBsAg gene subgenotype B3, cloning and transformation the HBsAg gene in Escherichia coli MC1061, and transformation in Lactococcus lactis (L. lactis). HBsAg gene subgenotype B3 was obtained from the pIDT-HBsAg subgenotype B3 plasmid. The HBsAg gene subgenotype B3 successfully cloned and transformed into E. coli MC1061 and L. lactis. The PCR results of the transformant E. coli MC1061 (pNZ8148-HBsAg subgenotype B3) colonies were found in colonies 8, 17, and 20 indicated by the presence of 1226 bps bands. 8 colonies were obtained from PCR results of L. lactis transformants (pNZ8148-HBsAg subgenotype B3). The construction of the HBsAg subgenotype B3 gene has 100% similarity compare to the hepatitis B virus isolated from Java on 1839. Therefore, the construction of pNZ8148-HBsAg subgenotype B3 using host cells L. lactis can be used as a vaccine candidate.

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The Expression of Codon Optimised Hepatitis B Core Antigen (HBcAg) of Subgenotype B3 Open Reading Frame in Lactococcus lactis

Cited by 2 publications

References 18 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

Genetic construction of HBsAg gene subgenotype B3 in Lactococcus lactis as hepatitis B vaccine candidate

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