• IMMUNE NETWORK
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• v.10 no.2
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• pp.35-45
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• 2010

Background: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. Methods: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize $V_H$ and $V_L$ fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. Results: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of $V_H$ or $V_L$ domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of VH of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. Conclusion: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.

• Parasites, Hosts and Diseases
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• v.46 no.3
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• pp.195-198
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• 2008

Plasmodium falciparum liver stage antigen-1 (PfLSA-1) is one of the few antigens expressed exclusively in liver stage parasites. In this study, we evaluated the antibody responses against recombinant PfLSA-1 in naturally infected individuals in Myanmar. High levels of antibody responses (70.7%) were detected in 82 serum samples from 116 infected individuals, and IgG responses to PfLSA-1 principally composed of responses of IgG1 and IgG3 subclasses. These results show that PfLSA-1 elicits effective antibody responses in individuals infected with P. falciparum, and thus it could be not only an attractive candidate protein for vaccine development, but also a useful antigen for serodiagnosis of the infection.

• Nuclear Medicine and Molecular Imaging
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• v.40 no.4
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• pp.211-217
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• 2006

Purpose: Recombinant ScFv lym-1 was produced, using pET vector system for large scale production. Methods: ScFv lym-1 gene inserted pET-22b (+) vector, was expressed in E.coli BL-21 strain. ScFv lym-1 antibody extracted from periplasm, was purified with His-Taq column. To evaluated immunoreactivity with Raji cell, ScFv lym-1 was labeled with I-125 and I-125 ScFv lym-1 was purified with desalting column. Raji cell was injected into the C57BR/cdJ SCID mice. Gamma camera imaging were taken time point at 1, 8, 24, and 48 hr with 8 mm pinhole collimator. Results: An active scFv lym-1 could be produced in E. coli with soluble iron using PET vector system. Immuuoreaetivity and affinity constant of IgG lym-1 were 54% and $1.83{\times}10^9M^{-1}$, respectively, and those of scFv lym-1 were 53.7% and $1.46{\times}10^9M^{-1}$, respectively. Biodistribution of I-125 scFv lym-1 antibody showed faster clearance in blood, spleen, kidney and than I-125 IgG lym-1 antibody. Gamma camera image of I-125 scFv lym-1 antibody showed faster clearance and tumor targeting liver than I-125 IgG lym-1 antibody. Conclusions: In vitro properties of scFv lym-1 were similar to those of IgG lym-1. ScFv lym-1 showed faster blood clearance than IgG lym-1 There results suggest that scFv lym-1 antibody can be useful for tumor imaging agent.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.82-82
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• 1997

The hepatitis B virus(HBV) is a small, enveloped virus with a circular, double-stranded DNA genome. HBV causes active and chronic hepatitis worldwide, including Korea, and is considered to be a major factor for liver cirrhosis and hepatocellular carcinoma. In contrast to the wealth of knowledge on the gene structure and expressional regulation, immunological and pathological mechanisms for HBV-induced hepatocellular injury are not well known. In the present study, vaccinia virus which has been demonstrated to be a useful eukaryotic expression vector was used to clone the gene for HBV surface antigen, L(S+preS2+preS1). The recombinant vaccinia virus vector, pMJ-L, which contains L surface antigen gene of adr-type HBV was constructed, and subseouently used for making recombinant vaccinia virus VV-$\textrm{HBV}_{L}$. Expression of the HBV antigen was examined by immunofluorescent antibody (IFA) test using mouse monoclonal anti-hepatitis B surface antigen. HBsAg was detected in the recombinant virus indicating that the VV-$\textrm{HBV}_{L}$ expressed S antigen successfully. The HBV-Vaccinia Virus recombinant obtained in this study is currently being used for studying the immunological aspects of HBV infection.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.712-720
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• 2007

To investigate clonal variations of recombinant Chinese hamster ovary(rCHO) clones in response to culture pH and temperature, serum-free suspension cultures of two antibody-producing CHO clones(clones A and B), which were isolated from the same parental clone by the limiting dilution method, were performed in a bioreactor at pH values in the range of 6.8-7.6, and two different temperatures, $33^{\circ}C\;and\;37^{\circ}C$. In regard to cell growth, clone A and clone B displayed similar responses to temperature, although their degree of response differed. In contrast, clones A and B displayed different responses to temperature in regard to antibody production. In the case of clone A, no significant increase in maximum antibody concentration was achieved by lowering the culture temperature. The maximum antibody concentration obtained at $33^{\circ}C$(pH 7.4) and $37^{\circ}C$(pH 7.0) were $82.0{\pm}2.6$ and $73.2{\pm}4.1{\mu}g/ml$, respectively. On the other hand, in the case of clone B, an approximately 2.5-fold increase in maximum antibody concentration was achieved by lowering the culture temperature. The enhanced maximum antibody concentration of clone B at $33^{\circ}C$($132.6{\pm}14.9{\mu}g/ml$ at pH 7.2) was due to not only enhanced specific antibody productivity but also to prolonged culture longevity. At $33^{\circ}C$, the culture longevity of clone A also improved, but not as much as that of clone B. Taken together, CHO clones derived from the same parental clone displayed quite different responses to culture temperature and pH with regards antibody production, suggesting that environmental parameters such as temperature and pH should be optimized for each CHO clone.

• Bulletin of the Korean Chemical Society
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• v.30 no.5
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• pp.1101-1106
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• 2009

Multivalent and multi-specific antibodies can provide valuable tools for bio-medical research, diagnosis and therapy. In antigen-antibody interactions, the avidity of antibodies depends on the affinity and the number of binding sites.$^1$ As artificial multivalent antibody agents, single chain Fv-streptavidin fusion tetramer proteins $(scFv-SA)_4$ have been previously tested.$^{1,\;2}$ Although, the Fab domain is known to be more stable than scFv in animal models,$^{3,\;4}$ it has never been used to make a multivalent agent with a streptavidin fusion. In this study, we prepared tetra-valent $(Fab-cSA)_4$ by fusing Fab with core streptavidin (cSA). This molecule was made using inclusion body production, refolding and chromatography purification. Affinities of the Fab-cSA tetramer and a scFv-cSA tetramer to a cell surface antigen were compared by ELISA using biotin-HRP. The Fab-cSA tetramer showed higher binding avidity than the scFv-cSA tetramer. The higher binding avidity of the Fab-cSA tetramer demonstrates its potential as a therapeutic agent for target-specific antibody therapy.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.916-920
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• 2002

This report describes a high-level expression of human alpha-2a interferon ($IFN{\alpha}-2a$) in Escherichia coli and its pilot scale purification by using a monoclonal antibody-independent chromatographic procedure that is based on anion-exchange, cation-exchange, hydrophobic interaction, and gel filtration. The recombinant E. coli produced much more $IFN{\alpha}-2a$ in a soluble form, when cultivated at low temperatures than at high-temperature fermentation. However, if the bacterial growth was taken into consideration, fermentation at $30^{\circ}C$ seemed optimal for the interferon production. By using our new protocol, we recovered approximately 160 mg of $IFN{\alpha}-2a$ with a specific activity of $3.59{\times}10^8$ IU/mg from 201 of the broth. The gel permeation chromatographic and SDS-PAGE indicated that the interferon preparation was purified to homogeneity and was of the correctly folded fast-migrating monomer.

• Korean Journal of Veterinary Research
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• v.47 no.1
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• pp.59-65
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• 2007

Pasteurella multocida Pasteurella multocida toxin (PMT) is a causal pathogenin atrophic rhinitis in pigs. To investigate the protective immunity and vaccination effect of recombinantPMT, the gene for PMT was isolated from the infective P. multocida D:4. The 2.3 kb XhoI/PstI fragment(PMT2.3) of PMT gene was expressed in E. coli BL21 (DE3) using the induced expression vector system.The recombinant protein of PMT2.3 having molecular weight of 84 kDa was purified by Ni-afinitycolumn chromatography. The PMT2.3 raised slightly less anti-PMT antibody titer than formalin-killedwhole cel, however, it showed more protective imunity against P. multocida D:4 infection in vaccinationand chalenge.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.403-403
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• 2005

• IMMUNE NETWORK
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• v.4 no.1
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• pp.23-30
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• 2004

Background: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah-1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating $\beta$-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. Conclusion: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.