• Nuclear Medicine and Molecular Imaging
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• v.43 no.5
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• pp.487-494
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• 2009

Purpose: Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation. Materials and Methods: Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FIT( conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt's lymphoma cells. Results: Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt's lymphoma cells. Conclusion: Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity.

• Journal of fish pathology
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• v.20 no.2
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• pp.119-127
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• 2007

Phosphoinositide-specific phospholipase Cδ (PLCδ) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to characterize the catalytic and regulatory properties of the PLCδ of Misgurnus mizolepis (ML-PLCδ). The ML-PLCδ gene was cloned and expressed under according to the method of the previous report (Kim et al., 2004), and its recombinant protein was purified by successive chromatography using Ni2+-NTA affinity column. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol (PI). Its activity was absolutely Ca2+-dependence, which was similar to mammalian PLCδ isozymes. The Ca2+ concentration yielding maximal activation of ML-PLCδ was 100 μM. However, the activity was decreased interestingly by a polyamine, such as spermine and spermidine. In vitro assay using cholate-micelle cell, ML-PLCδ activity was inhibited in dose-dependent manner by sphinogosine but increased by phosphocholine . In the lipid-binding assay, ML-PLCδ was strongly bound to LPA, PI(3)P, PI(4)P, PI(5)P, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3 and PA, but it showed the low affinity to S1P, PI(3,4)P2 and PS. Taken together our results, it is suggested that the general catalytic and regulatory properties of ML-PLCδ are similar with those of mammalian PLCδ1 isozymes, but the N-terminal extended piscine phospholipase Cδ1 (ML-PLCδ) might reflect some distinctions in regulatory properties and inositol-lipid binding specificity between piscine ML-PLCδ and mammalian PLCδ isozymes.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.274-280
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• 2005

Pasteurella multocida is known to cause widespread infections in husbandry. To induce homologous and heterologous immunity against the infections, outer membrane proteins (OMPs) in the envelope of P. multocida are thought to be attractive vaccine candidates. Outer membrane protein H is considered as the major component of OMPs. In this study, a gene for OmpH was isolated from pathogenic P. multocida serogroup A. The gene was composed of 1,047 nucleotides coding 348 amino acids with signal peptide of 20 amino acids. The amino acid composition showed about 80 to 98 per cent sequence homologies among other 10 strains of P. multocida serogroup A, reported so far. A recombinant ompH, from which signal peptide was truncated, was generated using pRSET A to name 'pRSET A/OmpH-F2'. The pRSET A/OmpH-F2 was well expressed in E. coli BL21(DE3). The truncated OmpH was purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Its molecular weight was registered to be 40 kDa on SDS-PAGE gel. In order to generate immunesera against the OmpH, 50 ug of the protein was intraperitoneally injected into mice three times. The anti-OmpH immuneserum recognized about $5{\times}10^{-2}$ng quantity of the purified OmpH. It can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (Serogroup A).

• Microbiology and Biotechnology Letters
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• v.40 no.2
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• pp.104-110
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• 2012

Glycosynthase is an active site nucleophile mutant enzyme, prepared from glycosidase, which is capable of synthesizing oligosaccharide derivatives without the hydrolysis of the product. Thermoacidophilic ${\alpha}$-glucosidase of Thermoplasma acidophilum (AglA) exhibits a transglycosylating activity yielding various glycosides. AglA was converted to glycosynthase by the substitution of the catalytic nucleophile Asp-408 residue into non-nucleophile glycine in order to increase its ability to synthesize various glycosides by transglycosylation. The glycosynthase mutant was purified by Ni-NTA chromatography and its glycoside-synthesizing activity was measured by using an external nucleophile, sodium formate buffer, providing maltose as a donor and p-nitrophenyl-${\alpha}$-D-glucopyranoside ($pNP{\alpha}G$) as an acceptor, respectively. In addition, $pNP{\alpha}G$ was examined for its feasibility to act as both a donor and an acceptor, and products were compared with those of the wildtype enzyme. The mutant enzyme was found to catalyze the formation of a specific product from $pNP{\alpha}G$ with a yield of 42.5% without further hydrolysis, while the wild-type enzyme produced two $pNP{\alpha}G$ products at low yields. The results demonstrate the possibility of satisfactory yields for the reactions in the presence of small amounts of acceptor, and demonstrate that the high activity of the mutant, at pHs below neutrality, was applicable in the transfer of glucose from the natural donor.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.606-612
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• 2018

The enzyme xylose isomerase (E.C. 5.3.1.5, XI) is responsible for the conversion of an aldose to ketose, especially xylose to xylulose. Owing to the ability of XI to isomerize glucose to fructose, this enzyme is used in the food industry to prepare high-fructose corn syrup. Therefore, we studied the characteristics of XI from Anoxybacillus kamchatkensis G10, a thermophilic bacterium. First, the gene coding for XI (xylA) was inserted into the pET-21a(+) expression vector and the construct was transformed into the Escherichia coli competent cell BL21 (DE3). The expression of recombinant XI was induced in the absence of isopropyl-thio-${\beta}$-galactopyranoside and purified using Ni-NTA affinity chromatography. The optimum temperature of recombinant XI was $80^{\circ}C$ and measurement of the heat stability indicated that 55% of residual activity was maintained after 2 h incubation at $60^{\circ}C$. The optimum pH was found to be 7.5 in sodium phosphate buffer. Magnesium, manganese, and cobalt ions were found to increase the enzyme activity; manganese was the most effective. Additionally, recombinant XI was resistant to the presence of $Ca^{2+}$ and $Zn^{2+}$ ions. The kinetic properties, $K_m$ and $V_{max}$, were calculated as 81.44 mM and $2.237{\mu}mol/min/mg$, respectively. Through redundancy analysis, XI of A. kamchatkensis G10 was classified into a family containing type II XIs produced by the genera Geobacillus, Bacillus, and Thermotoga. These results suggested that the thermostable nature of XI of A. kamchatkensis G10 may be advantageous in industrial applications and food processing.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1529-1536
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• 2006

We cloned a gene of ompH(D:4) from pigs infected with P. multocida D:4 in Korea [16]. The gene is composed of 1,026 nucleotides coding 342 amino acids (aa) with a signal peptide of 20 aa (GenBank accession number AY603962). In this study, we analyzed the ability of the ompH(D:4) to induce protective immunity against a wild-type challenge in mice. To determine appropriate epitope(s) of the gene, one full and three different types of truncated genes of the ompH(D:4) were constructed by PCR using pET32a or pRSET B as vectors. They were named ompH(D:4)-F (1,026 bp [1-1026] encoding 342 aa), ompH(D:4)-t1 (693 bp [55-747] encoding 231 aa), ompH(D:4)-t2 (561 bp [187-747] encoding 187 aa), and ompH(D:4)-t3 (540 bp [487-1026] encoding 180 aa), respectively. The genes were successfully expressed in Escherichia coli BL21(DE3). Their gene products, polypeptides, OmpH(D:4)-F, -t1, -t2, and -t3, were purified individually using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Their $M_rs$ were determined to be 54.6, 29, 24, and 23.2 kDa, respectively, using SDS-PAGE. Antisera against the four kinds of polypeptides were generated in mice for protective immunity analyses. Some $50{\mu}g$ of the four kinds of polypeptides were individually provided intraperitoneally with mice (n=20) as immunogens. The titer of post-immunized antiserum revealed that it grew remarkably compared with pre-antiserum. The lethal dose of the wild-type pathogen was determined at $10{\mu}l$ of live P. multocida D:4 through direct intraperitoneal (IP) injection, into post-immune mice (n=5, three times). Some thirty days later, the lethal dose ($10{\mu}l$) of live pathogen was challenged into the immunized mouse groups [OmpH(D:4)-F, -t1, -t2, and -t3; n=20 each, two times] as well as positive and negative control groups. As compared within samples, the OmpH(D:4)-F-immunized groups showed lower immune ability than the OmpH(D:4)-t1, -t2, and -t3. The results show that the truncated-OmpH(D:4)-t1, -t2, and -t3 can be used for an effective vaccine candidate against swine atrophic rhinitis caused by pathogenic P. multocida (D:4) isolated in Korea.

• Microbiology and Biotechnology Letters
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• v.40 no.3
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• pp.274-277
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• 2012

Cytokinesis is the final stage of cell division, dividing one mother cell into two daughter cells. For the cutting of a plasma membrane during bacterial cytokinesis, a tubulin homolog FtsZ protein is recruited from the cytoplasm to the division site. FtsZ protein polymerizes in a GTP-dependent manner and its N-terminal domain has a GTPase activity. In this study, we have begun to characterize FtsZ from Staphylococcus aureus (SA). Full-length SA FtsZ was cloned into pRSFDuet-1 vector and the clone was transformed into a BL21 (DE3) star cell. The recombinant SA FtsZ protein was purified using Ni-NTA affinity chromatography and dialysis. Using a spectrofluorometer, we showed that SA FtsZ undergoes a GTP-dependant polymerization in vitro. The polymer of the SA FtsZ protein disappeared after a few minutes, suggesting that the polymer is degraded as the GTP is consumed. This assay system may well be applied for inhibitor screening targeting S. aureus FtsZ.

• Korean Journal of Microbiology
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• v.42 no.2
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• pp.142-148
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• 2006

In order to develop chitosanase for the production of chitosan oligosaccharides, a chitosanase-producing bacterium was isolated from the traditional fermented soybean, Meju, and identified as Bacillus amyloliquefaciene MJ-1. The cloned chitosanase gene, 825 bp in size, encoded a single peptide of 274 amino acids with a estimated molecular mass of 30.9 kDa. The deduced amino acid sequence showed significant homology with microbial chitosanases. The recombinant chitosanase was expressed in Escherichia coli upon induction with isopropyl-D-thiogalactopyranoside, and purified using $Ni^{2+}-NTA$ agarose column chromatography. The maximal activity of the recombinant chitosanase is at pH 5.0 and $60^{\circ}C$. The recombinant chitosanase is stable between pH 5.0 and pH 7.0 at $37^{\circ}C$ for 30 min, and more than 75% of the activity still remain at $80^{\circ}C$ for 30 min incubation.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.355-362
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• 2016

The phosphomannomutase/phosphoglucomutase gene (pmm/pgm) of Vibrio anguillarum (the causative agent of fish vibriosis) was cloned, and the open reading frame corresponded to a protein with 446 amino acids. The pmm/pgm gene showed a significant degree of sequence homology with the previously reported genes from V. mimicus, V. vulnificus, V. splendidus, and V. harveyi, with 92.3%, 91.4%, 89.9%, and 89.9% amino acid identity, respectively. By reverse transcriptase-polymerase chain reaction, we found that the pmm/pgm gene was upregulated under cold stress condition. The PMM/PGM protein is known to catalyze the interconversion between mannose-1-phosphate and mannose-6-phosphate or glucose-1-phosphate and glucose-6-phosphate, which are important intermediates for lipopolysaccharide (LPS) biosynthesis. To confirm the role of PMM/PGM in the LPS biosynthetic pathway, we constructed a knock out mutant by homologous recombination. The respective LPSs were isolated from the V. anguillarum wild-type and mutant strains, and changes were compared by subjecting them to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on the different patterns of the LPSs, we expect the pmm/pgm gene to have an important role in LPS biosynthesis. The pmm/pgm-deficient mutant of V. anguillarum will contribute to further studies about the role of LPS in V. anguillarum pathogenesis.