• BMB Reports
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• v.33 no.2
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• pp.120-125
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• 2000

M2PI is an active single chain mini-proinsulin with a 9-residue linker containing the turn-forming sequence 'YPGDV' between the B- and A-chains, but which retains about 50% of native insulin receptor binding activity. The refolding efficiency of M2PI is higher than proinsulin by 20-40% at alkaline pH, and native insulin is generated by the enzymatic conversion of M2PI. The solution structure of M2PI was determined by NMR spectroscopy. The global structure of M2PI is similar to that of native insulin, but the flexible linker between the B- and A-chains perturbed the N-terminal A-chain and C-terminal B-chain. The helix in the N-terminal A-chain is partly perturbed and the ${\beta}$-turn in the B-chain is disrupted in M2PI. However, the linker between the two chains was completely disordered indicating that the designed turn was not formed under the experimental conditions (20% acetic acid). Considering the fact that an insulin analogue, directly cross-linked between the C-terminus of the B-chain and the N-terminus of the A-chain, has negligible binding activity, a flexible linker between the two chains is sufficient to keep binding activity of M2PI, but the perturbed secondary structures are detrimental to receptor binding.

• Archives of Pharmacal Research
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• v.27 no.4
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• pp.371-375
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• 2004

The silicon linker is the foremost traceless linker used in solid-phase reactions. Hydrogen fluo-ride (HF) or trifluoroacetic aicd (TFA) can remove the silicon linker with the silicon atom being replaced by a hydrogen atom. In this experiment, the linkers 1c and 2d, which are the most useful in solid-phase reactions, were synthesized, Linker 1c is composed of seven linearly linked carbons and linker 2d includes an oxygen atom in the linear carbon chain to increase the solvation capacity. The carboxylic acid component of linker 1c and 2d forms an amide or ester bond with resin. The synthesized linkers 1c and 2d could be utilized in constructing a chemical compound library that includes indole, benzodiazepine and phenothiazine (aromatic ring compounds).

• Archives of Pharmacal Research
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• v.26 no.10
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• pp.785-795
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• 2003

For the development of new anticonvulsive agents, GABAmimetics such as nipecotic acid, isonipecotic acid, ${\gamma}-aminobutyric$ acid (GABA), ${\gamma}-vinyl$ GABA (vigabatrin) and valproic acid were covalently coupled through an ester bond by a two-carbon linker chain as potential prodrugs and evaluated for their anticonvulsive activities.

• Journal of the Korean Society of Tobacco Science
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• v.37 no.1
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• pp.25-33
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• 2015

Genome walking is a par ticular application for identifying sequences of unknown genomic regions adjacent to a known region. Many genome walking methods based on polymerase chain reaction (PCR) are available. Even if earlier techniques suffer from low reproducibility, inefficiency, and non-specificity, improved strategies have been developed. In this study, we present an alternative strategy: the genomic DNA is digested with restriction enzymes. After cytosine overhangs at 5' ends, the fragments are ligated to linker adaptor s had guanine overhang at 3' ends. Then nested PCR is performed. The improvements in this strategy focus on two points. The first is the C tailing method using Pfu polymerase instead of the A tailing method based on nontemplate-dependent terminal transferase activity of Taq polymerase. Therefore unintended modification of target DNA can be prevented without A tailing error. The second point is the use of C/G-specific ligation had advantage in the ligation efficiency compared with A/T-specific ligation. Therefore, the C-G linker PCR method increases ligation efficiency between digested genomic DNA and adaptor DNA. As a result, the quantity of target DNA to amplify by PCR is enriched. We successfully used G-C linker PCR to retrieve flanking regions bordering the phophinothricin resistance gene in genetically modified tobacco (GMO).

• BMB Reports
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• v.30 no.3
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• pp.177-181
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• 1997

We constructed a single-chain immunoglobulin in which the carboxyl end of the heavy chain variable domain is covalently joined to the amino terminus of the light chain variable domain via peptide linker and the carboxyl end of the light chain variable domain is linked to human ${\gamma}1$ Fc region through the hinge region. The molecule was expressed in Chinese hamster ovary cells, assembled into a dimeric molecule and secreted into the culture medium. The dimeric molecule (2E11) was purified from the culture supernatant by affinity chromatography on Protein G-Sepharose column. The size of the unreduced or reduced protein was the expected molecular weight of approximately 120 or 60 kDa, respectively, as assessed by SDS-polyacrylamide gel electrophoresis. The antigen-binding affinity of 2E11 was almost the same as that of a native antibody counterpart (CS131A), suggesting that the single-chain immunoglobulin may function like a native antibody.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.197-206
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• 2003

hFSH (human follicle stimulating hormone) is heterodimer consisting of $\alpha$ and $\beta$ subunits. Since assembly of the both subunits in the cell is often the rate-limiting step in production of functional hormone, single-chain hormones have been engineered by genetically linking two different cDNA fragments with a linker sequence. Using retrovirus vector system, the resulting recombinant hFSH gene was transferred in CHO cells and chicken embryos, and the expression of the gene was investigated. In CHO cells, protein synthesis from the single-chain FSH gene was 17 fold higher than that from the heterodimeric counterpart. In the study of transgenic chickens, ten of the eleven chicks hatched from 62 embryos manupulated with recombinant retrovirus stock was determined to carry transgenic genes. RT-PCR analyses confirmed transcription of the single-chain FSH gene, however, no recombinant FSH was detected from the blood samples.

• Bulletin of the Korean Chemical Society
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• v.32 no.4
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• pp.1133-1137
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• 2011

Ordered array of gold nanoparticles (Au NPs) over ITO glass was investigated in terms of ITO pretreatment, particle size, and diamines with different chain length. Owing to the indium-tin-oxide (ITO) layer coated on the glass, the substrate surface has a limited number of hydroxyl groups which can produce functionalized amine groups for Au binding, which resulted in the loosely-packed array of Au NPs on the ITO surface. Diamine ligand as a molecular linker was introduced to enhance the lateral binding of adjacent Au NPs immobilized on the amine-functionalized ITO glass, consequently leading to the densely-packed array of Au NPs over the ITO substrate. The molecular bridging effect was strengthened with the increase of chain length of diamines: C-12 > C-8. The packing density of small Au NPs (< 40 nm) was significantly increased with the increase of C-8 diamine, but large Au NPs (> 60 nm) did not produce densely-packed array on the ITO glass even for the dosage of C-12 diamine.

• 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.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.246.1-246.1
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• 2003

Hetero chain compounds have high possibilities of being good medicinal candidate because of their well-known medicinal activity and relatively low subtitled carbon. By constructing the method of making this compound library, this research has the purpose to create a new medicinal candidate materials based on an easy medicinal search. The first step is to construct an Indole library in a compounding process with the design of a linker connecting a solid-state resin and a substrate. (omitted)

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1186-1190
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• 2008

Single-chain Fv (scFv) antibody against c-Met is expected to be employed in clinical treatment or imaging of cancer cells owing to the important biological roles of c-Met in the proliferation of malignancies. Here, we show that the productivity of scFv against c-Met in Escherichia coli is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders (i.e., $V_L$-linker-$V_H$ and $V_H$-linker-$V_L$), expressed them in the cytoplasm of E. coli trx/gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with $V_H/V_L$ orientation was more than five times higher than that with $V_L/V_H$ format. Coexpression of DsbC enhanced the yield of soluble amounts of anti-c-Met scFv protein for both constructs. The purified scFv antibodies of the two different formats exhibited almost the same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with $V_H/V_L$ or $V_L/V_H$ formats and obtained similar results to the case of scFv antibodies.