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Singh, Bijay,Lee, Chang Beom,Park, Je Won,Sohng, Jae Kyung Oxford University Press 2012 Protein engineering, design & selection Vol.25 No.4
<P>Two similar genes, dnmL and rmbA in Streptomyces peucetius, which encode for glucose-1-phosphate (G-1-P) thymidylyltransferases were expressed in Escherichia coli under similar conditions. While RmbA was expressed in soluble form, DnmL was found as insoluble aggregates in inclusion bodies. The difference in expression of these similar proteins led to investigate into the amino acid sequences of these proteins by sequence alignment, hydrophobicity scale and homology modeling. These analyses showed that the two proteins are different only in the C-terminal sequences. Deletion of C-terminal sequence of DnmL increased the expression level of truncated DnmL. Substitution of C-terminal sequence of DnmL with RmbA also expressed the recombinant protein in soluble form. Finally, mutation of six amino acids in DnmL rendered the protein expressed in soluble form. These results suggested that the soluble expression of the thymidylyltransferases lies in the C-terminal sequences. In conclusion, these methods of protein engineering will be a rational tool for enhancing solubility of proteins expressed in E.coli.</P>
Singh, Bijay,Jiang, Tao,Kim, You-Kyoung,Kang, Sang-Kee,Choi, Yun-Jaie,Cho, Chong-Su American Scientific Publishers 2015 Journal of nanoscience and nanotechnology Vol.15 No.1
<P>Swine dysentery is a contagious mucohaemorrhagic colitis of pigs that is caused by anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently, an outer membrane lipoprotein of B. hyodysenteriae (BmpB) has been identified, and the mice or pigs immunized with a recombinant BmpB generated antibodies recognizing the native BmpB of B. hyodysenteriae. In this study, we cloned, expressed and purified BmpB protein from E. coli and used it as a vaccine candidate for oral delivery. The BmpB was encapsulated into the pH-sensitive and thiolated Eudragit microspheres (TEMS). The sizes of the microspheres ranged from 5-20 관. About 22-34% of BmpB were released from the BmpB-loaded TEMS within 24 h at stomach pH 2.0 whereas the release of BmpB from the BmpB-loaded TEMS was 35% in the first one hour and reached 81% within 24 h at intestinal pH 7.2. These data revealed that the BmpB could be protected in the harsh gastric condition. Mucoadhesive experiment in vitro showed that TEMS have high binding affinity with the mucin glycoproteins of porcine intestine. Finally, in vitro production of cytokines from immune cells treated with the BmpB-loaded TEMS suggested that the TEMS would be a promising approach for oral delivery of BmpB as vaccine candidate.</P>
Singh, Bijay,Maharjan, Sushila,Jiang, Tao,Kang, Sang-Kee,Choi, Yun-Jaie,Cho, Chong-Su American Chemical Society 2015 Molecular pharmaceutics Vol.12 No.11
<P>Orally ingested pathogens or antigens are taken up by microfold cells (M cells) in Peyer’s patches of intestine to initiate protective immunity against infections. However, the uptake of orally delivered protein antigens through M cells is very low due to lack of specificity of proteins toward M cells and degradation of proteins in the harsh environment of gastrointestinal (GI) tract. To overcome these limitations, here we developed a pH-sensitive and mucoadhesive vehicle of thiolated eudragit (TE) microparticles to transport an M cell-targeting peptide-fused model protein antigen. Particularly, TE prolonged the particles transit time through the GI tract and predominantly released the proteins in ileum where M cells are abundant. Thus, oral delivery of TE microparticulate antigens exhibited high transcytosis of antigens through M cells resulting in strong protective sIgA as well as systemic IgG antibody responses. Importantly, the delivery system not only induced CD4<SUP>+</SUP> T cell immune responses but also generated strong CD8<SUP>+</SUP> T cell responses with enhanced production of IFN-γ in spleen. Given that M cells are considered a promising target for oral vaccination, this study could provide a new combinatorial method for the development of M-cell-targeted mucosal vaccines.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/mpohbp/2015/mpohbp.2015.12.issue-11/acs.molpharmaceut.5b00265/production/images/medium/mp-2015-002654_0015.gif'></P>
Chitosan-based particulate systems for the delivery of mucosal vaccines against infectious diseases
Singh, Bijay,Maharjan, Sushila,Cho, Ki-Hyun,Cui, LianHua,Park, In-Kyu,Choi, Yun-Jaie,Cho, Chong-Su Elsevier 2018 International journal of biological macromolecules Vol.110 No.-
<P><B>Abstract</B></P> <P>Given that most pathogens enter the body at mucosal surfaces for infection and mucosal immune responses act as the first line of defense against the invading pathogens, mucosal vaccination is the most effective method to prevent infectious diseases. However, the development of mucosal vaccines requires an efficient antigen delivery system which should protect the antigens from physical elimination and enzymatic degradation, target mucosal inductive sites, and appropriately stimulate the mucosal and systemic immunity. Accordingly, polymeric particles have garnered much attention because the physicochemical properties of polymers can be adjusted to resolve the issues associated with mucosal vaccine delivery. Particularly, chitosan-based polymeric carriers are the most promising vehicles for mucosal vaccine delivery because chitosan is biodegradable, biocompatible and mucoadhesive in nature. Similarly, chitosan can be modified with chemical and biological molecules to develop delivery carriers for controlled or targeted therapy. Moreover, they can be converted to various formulations, such as solid, liquid and gel, with a wide range of particle sizes. In this review, we highlight and discuss advances in the development of chitosan-based particulate systems, specifically for the delivery of mucosal vaccines against infections.</P>
Bijay Singh,Chang-Beom Lee,Jae Kyung Sohng 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
The doxorubicin biosynthetic gene cluster in Streptomyces peucetius ATCC 27952 was found to contain a TDP-D-glucose 4,6-dehydratase gene, dnsM, putatively involved in the biosynthesis of 2,3,6-trideoxy-3-aminohexose, daunosamine. But the gene contains a frameshift in the DNA sequence that caused the premature termination of translation. In pursuit of another TDP-D-glucose dehydratase in S. peucetius, we discovered a homologue gene, rmbB, whose deduced product exhibit a high sequence similarity to a number of TDP-D-glucose 4,6-dehydratases. The gene was found to be located within a putative rhamnose biosynthetic gene cluster at another locus of the genome. The rmbB protein was verified to be functional by enzyme assay as it converted TDP-D-glucose into TDP-4-dehydro-6-deoxy-D-glucose. Inactivation of rmbB from S. peucetius genome abolished the production of doxorubicin. Consequently, rmbB was proved to be essential for the biosynthesis of doxorubicin.
Needle-Free Immunization with Chitosan-Based Systems
Singh, Bijay,Maharjan, Sushila,Sindurakar, Princy,Cho, Ki-Hyun,Choi, Yun-Jaie,Cho, Chong-Su MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.11
<P>Despite successful use, needle-based immunizations have several issues such as the risk of injuries and infections from the reuse of needles and syringes and the low patient compliance due to pain and fear of needles during immunization. In contrast, needle-free immunizations have several advantages including ease of administration, high level of patient compliance and the possibility of mass vaccination. Thus, there is an increasing interest on developing effective needle-free immunizations via cutaneous and mucosal approaches. Here, we discuss several methods of needle-free immunizations and provide insights into promising use of chitosan systems for successful immunization.</P>
Bijay Singh,Chang Beom Lee,Jin Cheol Yoo,Kwang Kyoung Liou,Jae Kyung Sohng 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1
Streptomyces peucetius ATCC 27952 contains two similar genes (1068 nucleotides), dnmL and rmbA, which encode for glucose-1-phosphate (G-1-P) thymidylyltransferases (69% identity and 86% similarity). When the genes were expressed in E. coli under similar conditions, rmbA was expressed in soluble form, and it showed G-1-P thymidylyltransferase activity. But, dnmL was expressed as inclusion bodies. Thorough comparative analysis of DnmL and RmbA by sequence alignment, hydrophobicity scale and homology modeling inferred that the expression of RmbA in soluble form is contributed to the helical structure formed by the high hydrophobicity content in C-terminal sequence of RmbA. Similarly, the formation of inclusion bodies of DnmL was attributed to a loop conformation attained by its C-terminal sequence. To test the significance of these sequences, the C-terminal sequences of DnmL was substituted with that of RmbA. The fusion DnmL was expressed in soluble form and it had similar G-1-P thymidylyltransferase activity as compared to RmbA. These observations demonstrated that the hydrophobic effect plays a significant role in protein folding or structure, and the solubility of the expressed proteins depends on the protein conformations. In conclusion, this method of protein engineering will be a rational tool for enhancing solubility of proteins expressed in E. coli.