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

( Apon Zaenal Mustopa ),( Sri Kartika Wijaya ),( Ratih Asmana Ningrum ),( Dian Fitria Agustiyanti ),( Lita Triratna ),( Wida Nurul Alfisyahrin ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 한국미생물·생명공학회지 Vol.47 No.3

Hepatitis B treatments using immune therapy are gaining interest because of the improvements in dendritic cell performance for antigen presentation, which induces an appropriate immune response and raises patient survival rates. This research aims to produce a significant amount of the HBcAg antigen, which can induce an immune response and have a curative effect on HBV infection. In this study, the HBV subgenotype B3 of the HBcAg gene was used, which is dominant in Indonesia. Further, Lactococcus lactis bacteria was used as the host because of its safety and tightly regulated protein expression. The codon usage for the HBcAg gene was optimized to improve protein expression in L. lactis, which is important because a codon is not random between species. The HBcAg gene is attached to a pNZ8148 plasmid and transformed into the L. lactis NZ3900 expression host. The results confirm that a positive protein band (21 kDa) in two fractions of purified HBcAg was recognized by both western blotting and dot blot hybridization, even if the HBcAg optimized codon has higher GC contents than that suggested for L. lactis expression. Overall, this research strengthens the broad use of L. lactis bacteria for any protein expression, including higher protein expression of codon optimized HBcAg gene compared to non-optimized genes. Furthermore, the improvement in the codon optimization of the HBcAg gene significantly increases the total protein expression by 10-20%, and the expression level of the codon optimized HBcAg increases 1.5 to 3.2-times that of the native HBcAg.

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 The Korean Society for Microbiology and Biotechnol 2019 한국미생물·생명공학회지 Vol.47 No.3

Hepatitis B treatments using immune therapy are gaining interest because of the improvements in dendritic cell performance for antigen presentation, which induces an appropriate immune response and raises patient survival rates. This research aims to produce a significant amount of the HBcAg antigen, which can induce an immune response and have a curative effect on HBV infection. In this study, the HBV subgenotype B3 of the HBcAg gene was used, which is dominant in Indonesia. Further, Lactococcus lactis bacteria was used as the host because of its safety and tightly regulated protein expression. The codon usage for the HBcAg gene was optimized to improve protein expression in L. lactis, which is important because a codon is not random between species. The HBcAg gene is attached to a pNZ8148 plasmid and transformed into the L. lactis NZ3900 expression host. The results confirm that a positive protein band (21 kDa) in two fractions of purified HBcAg was recognized by both western blotting and dot blot hybridization, even if the HBcAg optimized codon has higher GC contents than that suggested for L. lactis expression. Overall, this research strengthens the broad use of L. lactis bacteria for any protein expression, including higher protein expression of codon optimized HBcAg gene compared to non-optimized genes. Furthermore, the improvement in the codon optimization of the HBcAg gene significantly increases the total protein expression by 10-20%, and the expression level of the codon optimized HBcAg increases 1.5 to 3.2-times that of the native HBcAg.

Antibiofilm Activity and Binding Specificity of Polyclonal DNA Aptamers on Staphylococcus aureus and Escherichia coli

Kusumawati Arizah,Mustopa Apon Zaenal,Umami Rifqiyah Nur,Santoso Adi,Wibawan I Wayan Teguh,Setiyono Agus,Sudarwanto Mirnawati Bachrum 한국미생물·생명공학회 2022 한국미생물·생명공학회지 Vol.50 No.3

Aptamers are short, chemically synthesized, single-stranded DNA or RNA oligonucleotides that fold into unique three-dimensional structures. In this study, we aim to determine the antibiofilm activity and binding specificity of the six polyclonal DNA aptamers (S15K3, S15K4, S15K6, S15K13, S15K15, and S15K20) on Staphylococcus aureus BPA-12 and Escherichia coli EPEC 4. Aptamer S15K6 showed the highest percentage of antibiofilm activity against S. aureus BPA-12 (37.4%) as shown by the lowest OD570 value of 0.313. Aptamer S15K20 showed the highest percentage of antibiofilm activity against E. coli EPEC 4 (15.4%) as shown by the lowest OD570 value of 0.515. Aptamers S15K13 and S15K20 showed antibiofilm activities against both S. aureus BPA-12 and E. coli EPEC4, and thus potentially have broad reactivity. Furthermore, based on the binding capacity and Kd values from our previous study, the binding specificity assay of selected polyclonal DNA aptamers (S15K3 and S15K15) against S. aureus BPA-12, E. coli EPEC 4, S. aureus BPA-6, S. agalactiae, E. coli MHA-6, and Listeria monocytogenes were performed using qPCR. Aptamers S15K3 and S15K15 showed specific binding to S. aureus BPA-12, E. coli EPEC 4, S. aureus BPA-6, and S. agalactiae, but could not bind to E. coli MHA-6 and L. monocytogenes. Therefore, this study showed that the polyclonal DNA aptamers have antibiofilm activity and were able to bind to S. aureus BPA-12 and E. coli EPEC 4 bacteria.

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

( Lita Meilina ),( Sri Budiarti ),( Apon Zaenal Mustopa ),( Huda Shalahudin Darusman ),( Lita Triratna ),( Muhammad Ajietuta Nugraha ),( Muhammad Sabiq Bilhaq ),( Ratih Asmana Ningrum ) 한국미생물생명공학회(구 한국산업미생물학회) 2021 한국미생물·생명공학회지 Vol.49 No.1

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 Usp₄₅ 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-SPU_sp45-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 IC₅₀ 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, codon-optimized 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.

Recent advancements of nitric oxide-releasing hydrogels for wound dressing applications

Hasan Nurhasni,Jiafu Cao,Mustopa Apon Zaenal,Himawan Achmad,Umami Rifqiyah Nur,Ullah Muneeb,Wathoni Nasrul,유진욱 한국약제학회 2023 Journal of Pharmaceutical Investigation Vol.53 No.6

Background Hydrogels are three-dimensional (3D) polymer networks that can absorb significant volumes of water inside their interstices and continue connecting these interstices while maintaining an inflated network structure; this capability is due to their unusual 3D crosslinked polymer meshwork structure. Hydrogels are promising wound dressings, particularly for chronic wounds, owing to their wound-healing properties, such as flexibility, adhesion, moisture control, biocompatibility, and biodegradability. Nitric oxide (NO) is a small molecule that has been thoroughly investigated for its wound-healing activity. The diverse roles of NO in wound healing require its stable delivery to the wound site. Thus, hydrogels have been evaluated as promising scaffolds for storing and releasing NO in a controlled manner to promote and accelerate wound healing. Area covered This review sought to introduce the types of polymers used to prepare hydrogel-based wound dressings and the types of NO donors used as wound healing promoters. The preparation method of the hydrogels and their physical and chemical properties were presented herein, and recent studies on NO-releasing hydrogels for wound therapy were summarized and discussed. Selected hydrogels with unique characteristics and significant findings for wound healing were also emphasized. Expert opinion Owing to the importance of chronic wounds in healthcare, the development of functional materials that support proper and rapid wound healing is required. NO-releasing hydrogels can be employed for wound dressing applications owing to their controlled NO releasability and antibacterial and wound healing activities.