• Korean Journal of Microbiology
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• v.51 no.2
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• pp.115-125
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• 2015

A monoclonal antibody AP64 IgM binds to human acute nonlymphocytic leukemia (ANLL) cell line HL60 and also cross-reacts with the homologous antigen in a rat ANLL cell. This antibody mediated by complement, has leukemia a suppression effect. In this study, we generated a recombinant single-chain variable domain fragment (ScFv) which were derived from $V_H$ and $V_L$ cDNA of AP64 IgM-secreting hybridoma by RT-PCR. The two variable regions were joined with a single 15 amino acid linker $(G_4S)_3$. This recombinant ScFv was expressed as a single polypeptide chain from Escherichia coli BMH 71-18. The recombinant ScFv was purified by applying the periplasmic extract to $Ni^+$-NTA-agarose affinity column and detected with westernblot. The purified recombinant ScFv recognized a surface antigen (about 30 kDa) of HL60 cell line which is the same antigen detected by parental AP64 IgM. But the affinity of ScFv for a surface antigen of HL60 was lower than that of the parental AP64 IgM, which needs to be further improved. Overall, the recombinant ScFv specific to HL60 might be a useful bioreagent for either diagnostic or therapeutic purposes.

• Journal of Life Science
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• v.25 no.6
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• pp.631-637
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• 2015

Streptavidin, a protein produced by Streptomyces avidinii, strongly binds up to four molecules of vitamin H, d-biotin exhibiting the dissociation constant of about 10⁻¹⁵ M. This strong binding affinity has been applied for detection and characterization of numerous biological molecules suggesting expression and purification of functional streptavidin should be very useful for the application of this streptavidin-biotin interaction. To express a soluble streptavidin in Escherichia coli, We synthesized streptavidin genes and cloned into pET-22b plasmid, which uses T7 RNA polymerase/T7 promoter expression systems containing pelB leader for secretion into periplasmic space and six polyhistidine tags at C-terminus for purification of expressed proteins. Although streptavidin is toxic to Escherichia coli due to strong biotin binding property, streptavidin was expressed very sufficiently in a range of 10-20 mg/ml. In SDS-PAGE, the size of purified protein was shown as 17 kDa in denatured condition (boiling) and 68 kDa in native condition (without boiling) suggesting tetramerization of monomeric subunit by non-covalent association. Further analysis by size-exclusion chromatography supported streptavidin’s tetrameric structure as well. In addition, soluble streptavidin detected biotinylated proteins in westernblot indicating its functional activity to biotin. Taken these results together, it concluded that our simple expression system was able to show high yield, homotetrameric formation and biotin binding activity analogous to natural streptavidin.

• Journal of Convergence for Information Technology
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• v.10 no.7
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• pp.249-256
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• 2020

To investigate the natural cosmetic ingredients of Benincasa hispida seed extract on skin care, we measured anti-oxidant and anti-inflammatory, and whitening effect. DPPH free radical scavenging activity was increased in a dose-dependent manner. The total phenolic content was higher in methanol extract (22.42±0.002 mgGAE/g) than water extract (9.77±0.002 mgGAE/g). MTT assay was demonstrated that the seed extract did not have a cytotoxic effect in RAW264.7 and B16F10 cell lines. We also examined to find out the inhibitory activity on NO production and secretion of TNF-α cytokine in LPS-induced RAW264.7 cells. In B16F10 melanoma cells, the seed extract significantly suppressed α-MSH induced melanin synthesis. Furthermore, westernblot analysis revealed that methanol extract dramatically downregulated the expression level of MITF, TRP-1 and TRP-2. Taken together, the B. hispida seed extract posses anti-oxidant, anti-inflammatory and skin whitening activities, which might provided its functional efficacy in cosmetic materials.

• The Korean Journal of Food And Nutrition
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• v.17 no.1
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• pp.47-52
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• 2004

In this study, I attempted to develope the expression and purification system of human mammaglobin proteins in Escherichia coli and to produce anti-human mammaglobin rabbit antibody for the detection of human mammaglobin protein in the peripheral blood of breast cancer patients. Human mammaglobin gene was cloned and sequenced from m-RNAs purified from donated breast cancer tissues using RT-PCR. The cloned gene was inserted into pET30, pET22, and pET32 plasmid. The cloned gene in pET30 yields insoluble proteins which was difficult to purify from the cells extracts. The mammaglobin gene in pET32 was strongly expressed soluble proteins which were isolated using Ni-NTA affinity chromagraphy and DEAE-ion exchange chromatography, followed by enterokinase digestion of the purified proteins. The isolated proteins had enough purity to use as a antigen for the production of anti-mammaglobin antibody in rabbits. The polyclonal antibody produced against the isolated mammaglobin showed a specificity to mammaglobin after Westernblot immuno assay. In conclusion, the isolated mammaglobin protein and the anti-mammaglobin rabbit antibody may be used for diagnosis of breast cancer as well as development of anti-breast cancer drug.

• The Journal of the Korean bone and joint tumor society
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• v.15 no.2
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• pp.111-121
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• 2009

Background: Osteosarcoma is one of the most common primary malignant tumors of bone occurring mainly in children and adolescents. Although surgery combined with chemotherapy has markedly improved patient survival during the last years, the use of anticancer drugs is still associated with serious problem, such as the frequent acquisition of drug-resistant phenotypes and occurrence of "secondary malignancies". Several solid tumors display enhanced expression of matrix metalloproteinases (MMPs), and recently clinical trials have been initiated on MMP-inhibitors. On the other hand, bisphosphonates (BPs) are inhibitors of bone resorption, and widely used to treat osteoclast-mediated bone diseases. Also they appear to possess direct antitumor activity. Methods: One osteosarcoma cell line (U2OS) was treated with ibandronate (0, 0.1, 1, $10{\mu}M$) for 48 hours. Cell viabilities were determined using MTT assay, the mRNA levels of MMP-2 and MT1-MMP were detected by reverse-transcription polymerase chain reaction, the amount of MMP-2 and MT1-MMP protein were measured by Westernblot, the activities of MMP-2 were observed by Gelatin zymography, and Matrigel invasion assays were used to investigate the invasive potential of osteosarcoma cell lines before and after ibandronate treatment. Results: The invasiveness of U2OS cell line was reduced dose-dependently following 48 hour treatment of up to $10{\mu}M$ of the ibandronate at which concentration no cytotoxicity occurred. Furthermore, the gelatinolytic activities and protein and mRNA levels of MMP-2 and MT1-MMP were also suppressed by increasing ibandronate concentrations. Conclusion: Given that MMP-2 is instrumental in tumor cell invasion, it is very likely that the reduction in osteosarcoma cell invasion by ibandronate is a consequence, at least in part, of suppressed expression of both MMP-2 and MT1-MMP. Isolation of a molecule (s) responsible for the bisphosphonate inhibition of tumor cell invasion would pave the way for the development of a new generation of metastasis inhibitors.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.330-342
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• 2018

The Streptavidin and Biotin system has been studied most extensively as the high affinity non-covalent binding of Biotin to STR ($K_D=10^{-14}M$) and four Biotin binding sites in tetrameric Streptavidin makes this system useful for the production of multivalent antibody. For the application of this system, we cloned Streptavidin amplified from Streptomyces avidinii chromosome by PCR and fused to gene of hAY4 single-chain Fv antibody specific to death receptor 4 (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand. The hAY4 single-chain Fv antibody fused to Streptavidin expressed in Escherichia coli showed 43 kDa monomer in heated SDS-PAGE. However, this fusion protein shown in both non-heated SDS-PAGE and Size-exclusion chromatography exhibited 172 kDa as a tetramer suggesting that natural tetramerization of Streptavidin by non-covalent association induced hAY4 single-chain Fv tetramerization. This fusion protein retained a Biotin binding activity similar to natural Streptavidin as shown in Ouchterlony assay and ELISA. Death receptor 4 antigen binding activity of purified hAY4 single-chain Fv fused to Streptavidin was also confirmed by ELISA and Westernblot. In addition, surface plasmon resonance analysis showed 60-fold higher antigen binding affinity of the hAY4-STR than monomeric hAY4 ScFv due to tetramerization. In summary, hAY4 single-chain Fv fused to Streptavidin fusion protein was successfully expressed and purified as a soluble tetramer in E. coli and showed both Biotin and DR4 antigen binding activity suggesting possible production of bifunctional and tetrameric ScFv antibody.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.298-304
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• 2007

Salmonella is facultative intracellular pathogen that can survive and replicate in macrophages even though these cells are equipped with a plethora of anti-microbial mechanisms. To survive in this hostile intracellular environment, Salmonella has evolved numerous defense mechartisms. An approximately 20 kDa protein was detected as a stationary-phase specific protein band in cytosolic fraction. It was identified as a DNA binding protein in stationary phase (Dps) by analysis of MALDI-TOF assay. It has been known that Dps, the protein produced in the stationary phase of bacteria, allows DNA to form chromatin by binding to DNA nonspecifically and protects DNA from reactive oxidative species (ROS). For further study, Dps specific polyclonal antibodies were generated by injection of purified Dps protein into rabbit. To examine the Finfluence of several regulatory proteins in the expression dps gene, Dps protein level in various S. typhimurium mutants defecting regulatory proteins were investigated by Westernblot using Dps specific polyclonal antibodies.

• Tuberculosis and Respiratory Diseases
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• v.53 no.1
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• pp.36-45
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• 2002

Background : In mammals, the activity of antioxidant enzymes is increased in adult lung to adapt to hyperoxia. The increase of these activities is augmented in neonates and is known as an important mechanism of tolerance to high oxygen levels. Peroxiredoxin(Prx) is an abundant and ubiquitous intracellular antioxidant enzyme. Prx I and II are major cytosolic subtypes. The aim of this study was to examine th Prx I and II mRNA and protein expression levels in adult rat lungs and to compare then with those of neonatal rat lungs exposed to hyperoxia. Materials and Methods : Adult Sprague-Dawley rats and neonates that were delivered from timed pregnant Sprague-Dawley rat were randomly exposed to normoxia or hyperoxia. After exposure to high oxygen level for a set time, the bronchoalveolar lavage fluid and lung tissue were obtained. The Prx I and II protein expression levels were measured by western blot analysis using polyclonal rabbit anti-Prx I or anti-Prx II antibodies and the relative expression of the Prx I and Prx II per Actin protein were obtained as an internal standard. The Prx I and II mRNA expression levels were measured by northernblot analysis using Prx I and Prx II-specific cDNA prepared from pCRPrx I and pCRPrx II, and the relative Prx I and Prx II expression levels per Actin mRNA were obtained as an internal standard. Results : Hyperoxia induced some peak increase in the Prx I mRNA levels after 24 hour in adult rats. Interestingly, hyperoxia induced a marked increase of Prx I mRNA 24 hour in neonatal rats. However, hyperoxia did not induce an alteration in the expression of Prx II mRNA in both the adult and neonatal rat lungs. Hyperoxia did not induce an alteration in the expression of the Prx I and Prx II protein in both the adult and neonatal rat lungs. Hyperoxia did not induce an alteration in the amount of Prx I and Prx II protein all the times in the bronchoalveolar fluid of adult rats. Conclusion : Prx I and II is differently regulated by hyperoxia in adult and neonatal rat lung at the transcriptional level. The prominent upregulation of Prx I mRNA in neonates compared to those in adults by hyperoxia may be another mechanism of resistance to high oxygen levels in neonate.

• Parasites, Hosts and Diseases
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• v.33 no.3
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• pp.201-210
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• 1995

This study aims to assess the possible strain-dependent variations in detection of ToxopLosmn antigens and antibodies. The virulent RH strain or avirulent Beverley strain of T gondii were injected into mice, intraperitoneally, and their antigens, antibodies and parasites were identified from the blood or tissues: liver, brain and spleen by ELISA, Western blot and PCR. In mice infected with RH strain, circulating antigens and parasitemia were first detected from 2 days after infection, and ToxopIasma DNA were found in the blood, liver, brain and spleen from 3 days after infection. It was impossible to detect specific IgM and IgG antibodies to T gondij and any specific band was not found by Western blot. In mice infected with Beverley strain, circulating antigens were detected between day 10 and day 35. The Toxoplusma DNA was found in the blood and liver from day 15 until day 60, and in the brain from day 20. But Toxoplosma DNA in the spleen were mainly detected between day 10 and day 30. The IgM antibodies were first appeared on day 10 post-infection, and were noted obviously increased between day 15 and 25. The IgG antibodies were first detected on day 15, and showed progressively increased titers. The antibody binding bands were specific according to infection period. Sera from mice infected with Beverley strain reacted mainly with the antigen of 27.5-kDa and 32.5-kDa. In conclusion, mice infected with RH strain revealed Toxoplosma antigens strongly, but not antibodies. However. mice infected with Beverley strain revealed both the Toxoplasma antigens and antibodies. The present results showed that immune responses are different between avirulent and virulent T gonnii.

• Journal of Life Science
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• v.28 no.12
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• pp.1416-1423
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• 2018

With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.