• Journal of Life Science
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• v.14 no.5
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• pp.840-846
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• 2004

A study for expression of Korean Mistletoe (KM) lectin gene (A,B) in Saccharomyces cerevisiae was done using transforming system of yeast. In order to overexpress the genes efficiently in yeast, two lectin genes (A,B) were re-cloned and modified including Kozak translation initiation sequence using PCR amplification. The constructed plasmids containing modified lectin A and B genes were transformed to S. cerevisea INVSc (MAT G, his3 $\Delta$1, leu2, trpl-289, ura3-52). The transformed cells were identified by DNA sequencing with ABI3700 system and induced with 2% of galactose for recombinant KM lectin (rKM lectin) protein. The rKM lectin A and B proteins were determinated about 29kDa size of protein by SOS-P AGE and western blotting analysis. The expressed recombinant lectin was determinated 1.24∼1.75 $\mu\textrm{g}$ per 1 mg of cytosolic soluble protein by sandwich ELISA method. Moreover the lectin genes were expressed as maximum level at 36 h after galactose induction and lectin A gene was were repressed after 48 h.

• YAKHAK HOEJI
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• v.46 no.4
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• pp.270-275
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• 2002

The lectin gene from rice was amplified by the polymerase chain reaction. The amplified DNA was inserted into the expression vector pET26b and expressed it as a fusion protein with polyhistidine sequences in Escherichia coli. The recombinant protein was produced by induction with 0.4 mM isopropyl-$\beta$-D-thiogalactopyranoside at 37$^{\circ}C$ and purified by an immobilized metal affinity chromatography. The recombinant protein was found to have lectin activity by the hemagglutination inhibition assay. The hemagglutination activity of the recombinant protein was optimal at pH 4.0-7.0 and was dependent on $Ca^{2+}$ and Mn$^{2+}$.2+/.

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.1
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• pp.40-43
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• 1998

The glycosylation pattern of Erythropoietin (EPO), produced by recombinant CHO cells, was studied using the simple and rapid technique of 'Lectin-blotting'. In this experiment we used three different kinds of lectins, MAA(Maackia amurensis agglutinine), RCA(Ricinus communis agglutinine), and DSA(Datura stramonium agglutinine), which bind to the terminal sialic acid, galactose, and the N-acetyllactosamine chain respectively. The lectin-blotting technique was used to analyze the carbohydrate structure of EPO produced in the presence of two physiologically active chemical compounds, ammonium and chloroquine. The effect of the ammonium ion on the glycosylation of EPO was studied because it accumulated in the medium mainly as a by-product of glutamine matabolism. Ammonium chloride significantly inhibited the sialylation of the terminal galactose residue at concentrations of 8mM or more. Chloroquine, a potent inhibitor of glycosylation, inhibited terminal sialylation at concentrations of 100 and 200 $\mu$M, and at a concentration of 300 $\mu$M, also inhibited Nacetyllactosamine chain synthesis.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.547-554
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• 1997

Galectin-3 is known as an animal ${\beta}$-galactoside-binding lectin charicterized with S-type carbohydrate recognition domain. It plays a role in growth, adherence and movement of cells. It is, also, related to the cell transformation and metastasis of tumor cells. In this study, we have expressed and purified recombinant human galectin-3 (rHgalectin-3) using E coli system and asialofetuin affinity chromatography for the future development of monoclonal antibody to Hgalectin-3, which is suggested as the tumor marker for the gastric and thyroid gland cancers. Expressed protein was confirmed as the Hgalectin-3 by immunoblot with cross-reactive murine monoclonal antibody. Lectin activity and specificity of purified protein were, also, confirmed by the competitive inhibition with galectin-3 specific carbohydrate, lactose. Like physiological galectin-3, lectin activity of the molecule was not changed in nonreduced condition. Dimer formation, furthermore, was observed at high concentration of the protein even in the reduced condition, which is well known in physiological galectin-3. These results showed purified rHgalectin-3 has the same activity and molecular nature compared to the physiological galectin-3.

• Journal of Life Science
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• v.19 no.2
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• pp.284-288
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• 2009

The innate immune system is important for the first line of host defence against infectious agents, which have penetrated the mechanical barriers. Mannose-binding lectin (MBL or mannan-binding protein, MBP) is a serum protein that is synthesized in the liver as a part of the acute phase response. MBL binds to carbohydrate structures presented by a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL is synthesized as a monomer that has a carboxy-terminal carbohydrate recognition domain, a neck region and a collagen region. Low MBL level was reported to be the most frequent immuno-deficiency syndrome. Although extensive studies have yielded detailed information on the structure of MBL, functions of the MBL complex are not fully understood yet. We, here, present cloning process of MBL cDNA from the rat liver and production of truncated recombinant MBL protein using a bacterial expression system in order to produce anti-MBL polyclonal antibody. Anti-MBL polyclonal antibody was raised in a New Zealand rabbit and its affinity was tested against recombinant protein using western blot technique. MBL cDNA, recombinant protein and anti-MBL antibody could be used as great arsenals to dissect cellular biochemistry of MBL.

• BMB Reports
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• v.39 no.5
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• pp.560-570
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• 2006

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the A-chain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.

• Journal of Microbiology and Biotechnology
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• v.33 no.5
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• pp.698-705
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• 2023

Rapid diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for guiding clinical treatment and preventing the spread of MRSA infections. Herein, we present a simple and rapid MRSA screening test based on the aggregation effect of mannose-binding lectin (MBL)-conjugated gold nanoparticles (AuNP), called the MRSA probe. Recombinant MBL protein is a member of the lectin family and part of the innate immune system. It can recognize wall teichoic acid (WTA) on the membrane of MRSA more specifically than that of methicillin-sensitive Staphylococcus aureus (MSSA) under optimized salt conditions. Thus, the MRSA probe can selectively bind to MRSA, and the aggregation of the probes on the surface of the target bacteria can be detected and analyzed by the naked eye within 5 min. To demonstrate the suitability of the method for real-world application, we tested 40 clinical S. aureus isolates (including 20 MRSA specimens) and recorded a sensitivity of 100%. In conclusion, the MRSA probe-based screening test with its excellent sensitivity has the potential for successful application in the microbiology laboratory.

• Journal of Chest Surgery
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• v.45 no.6
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• pp.380-389
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• 2012

Background: Bovine pericardium is one of the most widely used materials in bioprosthetic heart valves. Immunologic responses have been implicated as potential causes of limited durability of xenogenic valves. This study aimed to determine the effectiveness of decellularization and ${\alpha}$-galactosidase (${\alpha}$-gal) to remove major xenoreactive antigens from xenogenic tissues. Materials and Methods: Recombinant Bacteroides thetaiotaomicron (B. thetaiotaomicron) ${\alpha}$-gal or decellularization, or both were used to remove ${\alpha}$-gal from bovine pericardium. It was confirmed by ${\alpha}$-gal-bovine serum albumin-based enzyme-linked immunosorbent assay (ELISA), high-performance anion exchange chromatography, flow cytometry, 3,3'-diaminobenzidine-staining, and lectin-based ELISA. The mechanical properties of bovine pericardium after decellularization or ${\alpha}$-gal treatment were investigated by tests of tensile-strength, permeability, and compliance. Collagen fiber rearrangement was also evaluated by a 20,000${\times}$ transmission electron microscope (TEM). Results: Recombinant B. thetaiotaomicron ${\alpha}$-gal could effectively remove ${\alpha}$-gal from bovine pericardium B. thetaiotaomicron (0.1 U/mL, pH 7.2) while recombinant human ${\alpha}$-gal removed it recombinant human ${\alpha}$-gal (10 U/mL, pH 5.0). There was no difference in the mechanical properties of fresh and recombinant ${\alpha}$-gal-treated bovine pericardium. Furthermore, the TEM findings demonstrated that recombinant ${\alpha}$-gal made no difference in the arrangement of collagen fiber bundles with decellularization. Conclusion: Recombinant B. thetaiotaomicron ${\alpha}$-gal effectively removed ${\alpha}$-gal from bovine pericardium with a small amount under physiological conditions compared to human recombinant ${\alpha}$-gal, which may alleviate the harmful xenoreactive immunologic responses of ${\alpha}$-gal. Recombinant ${\alpha}$-gal treatment had no adverse effects on the mechanical properties of bovine pericardium.

• BMB Reports
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• v.46 no.7
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• pp.358-363
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• 2013

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.383-391
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• 2008

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed in mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennarytype complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using a pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of Nacetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using $UDP-^{14}C-Gal\;and\;CMP-^{14}C-Sia$ as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the ed blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.