• 한국진공학회지
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• 제17권6호
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• pp.549-554
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• 2008

수소 플라즈마 처리된 유리 기판에 스핀 코팅 시스템을 이용하여 nickel chloride를 코팅하여 단백질칩 플레이트를 제조하였다. 다양한 플라즈마 처리 시간대에서 histidine tagged 단백질의 부착 능력 특성을 연구하였다. 유리 기판 표면에서 nickel chloride와 단백질 특성을 particle size analysis를 이용하여 관찰하였고, 단백질의 부착 능력 정도를 bio imaging analyzer system으로 측정하였다. 실험 결과에 따르면, 플라즈마 처리 시간이 증가할수록 단백질 부착 능력은 감소하는 것으로 나타났다. 기판 표면에서의 단백질 부착능력 특성에 관한 mechanism은 본문의 결과 및 토의에서 논의되었다. 플라즈마 처리된 단백질칩 기판에 대한 표면 안정화는 바이오센서 시장에서 큰 관심을 끌 것으로 기대된다.

• Genomics & Informatics
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• 제4권3호
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• pp.133-136
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• 2006

The adsorption of proteins on the surface of glass slides is essential for construction of protein chips. Previously, we prepared a nickel-coated plate by the spin-coating method for immobilization of His-tagged proteins. In order to know whether the structural factor is responsible for the immobilization of His-tagged proteins to the nickel-coated glass slide, we executed a series of experiments. First we purified a His-tagged protein after expressing the vector in E. coli BL21 (DE3). Then we obtained the unfolding curve for the His-tagged protein by using guanidine hydrochloride. Fractions unfolded were monitored by internal fluorescence spectroscopy. The ${\Delta}G_{H20}$ for unfolding was $2.27kcal/mol{/pm}0.52$. Then we tested if unfolded His-tagged proteins can be adsorbed to the nickel-coated plate, comparing with $Ni^{2+}-NTA$ (nitrilotriacetic acid) beads. Whereas unfolded His-tagged proteins were adsorbed to $Ni^{2+}-NTA$ beads, they did not bind to the nickel-coated plate. In conclusion, a structural factor is likely to be an important factor for constructing the protein chips, when His-tagged proteins will immobilize to the nickel-coated slides.

• Bulletin of the Korean Chemical Society
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• 제23권12호
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• pp.1724-1728
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• 2002

The adsorption of proteins on the surface of glass slides is essential for the construction of protein chips. Here, we report that a Histidine (His)-tagged protein protein has been efficiently adsorbed on glass coated with nickel. A variety of nickel chloride-coated plates were prepared by the spin-coating method and adsorbed to the His-tagged protein. When the protein was adsorbed onto the surface of a variety of nickel chloride-coated glass slides, the efficiency of protein adsorption was dependent upon the coating conditions such as nickel chloride concentration, the spin speed and the drying temperature. The slides appropriate for protein adsorption were obtained when the slides were coated with 11%(w/w) of $NiCl_2$ at the spin speed of 4000 rpm for 20 sec and then dried at higher than 40°C. The physical properties of their nickel chloride thin layer were characterized by scanning electron microscopy. x-ray diffraction and atomic force microscopy, finding that the nickel chloride particles were around 10 nm in diameter and uniformly crystallized at 101 faces. These results show that nickel chloride-coated slides prepared by the spin-coating method are utilizable for the construction of Histagged protein chips.

• 한국표면공학회지
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• 제37권2호
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• pp.76-79
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• 2004

Nickel chloride coated protein chip plate was developed by using a spin coating method. The ability of histidine tagged protein adsorption was investigated at various solvents. The surface of plate has a large aggregated nickel complex with high density in water. However, the surface of plate has a very small size of aggregated nickel complex with low density in isopropanol. The ability of protein adsorption decreased as increasing the size of alkyl chain in various alcohol solvents. The mechanism on the ability of protein adsorption at the plate surface is discussed.

• 한국축산식품학회지
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• 제39권4호
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• pp.601-609
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• 2019

Bifidobacterium longum KACC 91563 secretes family 5 extracellular solute-binding protein via extracellular vesicle. In our previous work, it was demonstrated that the protein effectively alleviated food allergy symptoms via mast cell specific apoptosis, and it has revealed a therapeutic potential of this protein in allergy treatment. In the present study, we cloned the gene encoding extracellular solute-binding protein of the strain into the histidine-tagged pET-28a(+) vector and transformed the resulting plasmid into the Escherichia coli strain BL21 (DE3). The histidine-tagged extracellular solute-binding protein expressed in the transformed cells was purified using Ni-NTA affinity column. To enhance the efficiency of the protein purification, three parameters were optimized; the host bacterial strain, the culturing and induction temperature, and the purification protocol. After the process, two liters of transformed culture produced 7.15 mg of the recombinant proteins. This is the first study describing the production of extracellular solute-binding protein of probiotic bacteria. Establishment of large-scale production strategy for the protein will further contribute to the development of functional foods and potential alternative treatments for allergies.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.29-36
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• 2007

The xynA gene encoding the xylanase A of Paenibacillus sp. DG-22 was isolated with a DNA probe obtained by PCR amplification, using degenerated primers deduced from the amino acid residues of the known N-terminal region of the purified enzyme and the conserved region in the family 11 xylanases. The positive clones were screened on the LB agar plates supplemented with xylan, by the Congo-red staining method. The xynA gene consists of a 630-bp open reading frame encoding a protein of 210 amino acids, and the XynA preprotein contains a 28-residues signal peptide whose cleavage yields a l82-residues mature protein of a calculated molecular weight of 20,000Da and pI value of 8.77. The cloned DNA fragment also has another ORF of 873 nucleotides that showed 76% identity to the putative transcriptional activator of Bacillus halodurans C-125. Most of the xylanase activity was found in the periplasmic space of E. coli. The xynA gene was subcloned into pQE60 expression vector to fuse with six histidine-tag. The recombinant xylanase A was purified by heating and immobilized metal affinity chromatography. The optimum pH and temperature of the purified enzyme were 6.0 and $60^{\circ}C$, respectively. This histidine-tagged xylanase A was less thermostable than the native enzyme.

• 한국표면공학회지
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• 제40권2호
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• pp.98-102
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• 2007

[ $NiCl_2$ ] and poly-L-lysine coated protein chip plates have been fabricated using a spin coating system. Water has been used as solvent and scratching effects on glass slides and ITO have been investigated. We also observed the surface properties of $NiCl_2$ and poly-L-lysine coated slides by using PSA(Particle size analyzer) and AFM(Atomic force microscope). The AFM results imply that the surface patterns created in the spin coating system determine the protein adsorption. Adsorption of histidine-tagged KRS proteins immobilized on glass slides and ITO was analyzed using a BAS image system. The results suggest that the scratching effect was increased ability of protein adsorption.

• 약학회지
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• 제47권6호
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• Biomolecules & Therapeutics
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• 제9권2호
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• pp.79-87
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• 2001

Heat shock proteins serve as chaperone by preventing the aggregation of denatured proteins and promote survival of pathogens in harsh environments. In this study, heat shock gene encoding a 84-kDa (p84) protein, which is one of the three major heat shock proteins in S. pneumoniae, was cloned and characterized. PCR with a forward primer derived from N-terminal amino acid sequence of the p84 and a reverse primer derived from the conserved second ATP-binding region of Clp family was used for amplification of the gene encoding the p84 and subsequently the PCR product was used for sequence determination. Sequence analysis of the p84 gene demonstrated that it is a member of ClpL. The deduced amino acid sequence of pneumococcal ClpL shows homology with other members of the Clp family, and particularly, even in variable leader region, with bovine Clp-like protein and L. lactis ClpL. S. pneumoniae clpL is the smallest clop member (701 amono acids) containing the two conserved ATP-binding regions, and hydrophilic N-terminal variable region of pneu-mococcal Clp ATPase is much shorter than any known Clp ATPases. Histidine tagged ClpL was overexpressed and purified from E. coli. Immunoblot analysis employing antisera raised against pneumococcus p84 demonstrated no cross-reactivity with Clp analog in Eschericha coli, Staphylococcus aureus and human HeLa cells. Preimmunization of mice with ClpL extended mice life partially but did not protect them from death.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1291-1299
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• 2014

Recently, multifunctional nanomaterials have been developed as nanotherapeutic agents for cellular imaging and targeted cancer treatment because of their ease of synthesis and low cytotoxicity. In this study, we developed a multifunctional, two-component nanorod consisting of gold (Au) and nickel (Ni) blocks that enables dual-fluorescence imaging and the targeted delivery of small interfering RNA (siRNA) to improve cancer treatment. Fluorescein isothiocyanate-labeled luteinizing hormone-releasing hormone (LHRH) peptides were attached to the surface of a Ni block via a histidine-tagged LHRH interaction to specifically bind to a breast cancer cell line, MCF-7. The Au block was modified with TAMRA-labeled thiolated siRNA in order to knock down the vascular endothelial growth factor protein to inhibit cancer growth. These two-component nanorods actively targeted and internalized into MCF-7 cells to induce apoptosis through RNA interference. This study demonstrates the feasibility of using two-component nanorods as a potential theranostic in breast cancer treatment, with capabilities in dual imaging and targeted gene delivery.