• BMB Reports
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• 제32권5호
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• pp.497-501
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• 1999

Biotinylation of recombinant proteins is a powerful tool for the detection and analysis of proteins of interest in a large variety of assay systems. The recent development of in vivo biotinylation techniques in E. coli has opened new possibilities for the production of site-specifically biotinylated proteins without the need for further manipulation after the isolation of the recombinantly expressed proteins. In the present study, a novel vector set was generated which allows the convenient cloning and expression of proteins of interest fused with an N-terminal in vivo biotinylated thioredoxin (TRX) protein. These vectors were derived from the previously reported pBIOTRX vector into which was incorporated part of the pBluescript II+phagemid multiple cloning site (MCS), amplified by PCR using a pair of sophisticated oligonucleotide primers. The functionality of these novel vectors was examined in this system by recombinant expression of rat transforming growth factor-$\beta$. Western-blot analysis using TRX-specific antibodies or peroxidase-conjugated streptavidin confirmed the successful induction of the fusion protein and the in vivo conjugation of biotin molecules, respectively. The convenience of molecular subcloning provided by the MCS and the effective in vivo biotinylation of proteins of interest makes this novel vector set an interesting alternative for the production of biotinylated proteins.

• Genomics & Informatics
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• 제20권2호
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• pp.24.1-24.8
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• 2022

Hypomyelinating leukodystrophy type 2 (HLD2), is an inherited genetic disease of the central nervous system caused by recessive mutations in the gap junction protein gamma 2 (GJC2/GJA12). HLD2 is characterized by nystagmus, developmental delay, motor impairments, ataxia, severe speech problem, and hypomyelination in the brain. The GJC2 sequence encodes connexin 47 protein (Cx47). Connexins are a group of membrane proteins that oligomerize to construct gap junctions protein. In the present study, a novel missense mutation gene c.760G>A (p.Val254Met) was identified in a patient with HLD2 by performing whole exome sequencing. Following the discovery of the new mutation in the proband, we used Sanger sequencing to analyze his affected sibling and parents. Sanger sequencing verified homozygosity of the mutation in the proband and his affected sibling. The autosomal recessive inheritance pattern was confirmed since Sanger sequencing revealed both healthy parents were heterozygous for the mutation. PolyPhen2, SIFT, PROVEAN, and CADD were used to evaluate the function prediction scores of detected mutations. Cx47 is essential for oligodendrocyte function, including adequate myelination and myelin maintenance in humans. Novel mutation p.Val254Met is located in the second extracellular domain of Cx47, both extracellular loops are highly conserved and probably induce intramolecular disulfide interactions. This novel mutation in the Cx47 gene causes oligodendrocyte dysfunction and HLD2 disorder.

• 한국미생물·생명공학회지
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• 제49권4호
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• pp.510-518
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• 2021

Bacteriophages are considered excellent sensing elements for platforms detecting bacteria. However, their lytic cycle has restricted their efficacy. Here, we used the minor coat protein pIII domain (N1N2) of phage CTXφ to construct a novel surface plasmon resonance (SPR) biosensor that could detect Vibrio cholerae. N1N2 harboring the domains required for phage adsorption and entry was obtained from Escherichia coli using recombinant protein expression and purification. SDS-PAGE revealed an approximate size of 30 kDa for N1N2. Dot blot and transmission electron microscopy analyses revealed that the protein bound to the host V. cholerae but not to non-host E. coli K-12 cells. Next, we used amine-coupling to develop a novel recombinant N1N2 (rN1N2)-functionalized SPR biosensor by immobilizing rN1N2 proteins on gold substrates and using SPR to monitor the binding kinetics of the proteins with target bacteria. We observed rapid detection of V. cholerae in the range of approximately 10³ to 10⁹ CFU/ml but not of E. coli at any tested concentration, thereby confirming that the biosensor exhibited differential recognition and binding. The results indicate that the novel biosensor can rapidly monitor a target pathogenic microorganism in the environment and is very useful for monitoring food safety and facilitating early disease prevention.

• 대한방사성의약품학회지
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• 제7권1호
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• pp.56-65
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• 2021

TSPO, an 18-kDa translocator protein, is a peripheral-type benzodiazepine receptor that has been associated to a variety of biological activities such as apoptosis, steroidogenesis, and cell proliferation. Because TSPO overexpression has been found in various forms of cancer, it has recently become one of the most appealing biological targets for cancer therapies and detection. In order to create new optical imaging agents for improved diagnostics, we synthesized a novel dimeric fluorescent TSPO ligand based on PRB28 structure and SCy5.5. Following the preparation of the novel TSPO ligand, in vivo and ex vivo imaging tests were performed to examine the tumor uptake characteristics of the fluorescent TSPO ligand in a glioma animal model, and it was found that novel TSPO ligand was accumulated in glioma. These results suggested that novel dimeric fluorescent TSPO ligand will be applied to detect glioma.

• BMB Reports
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• 제37권2호
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• pp.159-166
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• 2004

Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of $3.4{\times}10^{-7}\;M$. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.

• 한국정보통신학회논문지
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• 제18권8호
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• pp.2017-2022
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• 2014

폴리히드론 프로모터, 수포성구내염 바이러스G, 폴리A, 사이토메가바이러스 프로모터, 강화녹색형광단백질, 단백질전달부위 유전자 등을 포함한 새로운 베큘로바이러스 시스템이 제조되었다. 본 재조합벡터 시스템은 인간 섬유아세포에 적용하여 시험하였고 재조합된 유전자의 전달과 유전자 발현을 대조 벡터시스템과 비교하였다. 본 연구로부터 새롭게 제작된 본 베큘로바이러스 시스템이 유전자의 전이와 유전자 발현 면에서 대조 벡터시스템 보다 고효율을 나타내었다.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.408.2-409
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• 2002

The aim of this study is to prepare biodegradable microspheres without use of any kind of surfactants or emulsifiers for a novel sustained delivery carriers of protein drugs. Poly(e-caprolactone)/poly(ethylene glycol)/poly(e-caprolactone) (CEC) triblock copolymer was synthesized by ring-opening of e-caprolactone with dihydroxy poly(ethylene glycol) and was used to make surfactant-free microspheres. (omitted)

• BMB Reports
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• 제49권11호
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• pp.585-586
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• 2016

Extracellular vesicles (EVs) are natural carriers of biomolecules that play central roles in cell-to-cell communications. Based on this, there have been various attempts to use EVs as therapeutic drug carriers. From chemical reagents to nucleic acids, various macromolecules were successfully loaded into EVs; however, loading of proteins with high molecular weight has been huddled with several problems. Purification of recombinant proteins is expensive and time consuming, and easily results in modification of proteins due to physical or chemical forces. Also, the loading efficiency of conventional methods is too low for most proteins. We have recently proposed a new method, the so-called exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs), to overcome the limitations. Since EXPLORs are produced by actively loading of intracellular proteins into EVs using blue light without protein purification steps, we demonstrated that the EXPLOR technique significantly improves the loading and delivery efficiency of therapeutic proteins. In further in vitro and in vivo experiments, we demonstrate the potential of EXPLOR technology as a novel platform for biopharmaceuticals, by successful delivery of several functional proteins such as Cre recombinase, into the target cells.

• Molecules and Cells
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• 제27권6호
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• pp.629-634
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• 2009

G protein-coupled receptors (GPCRs) are part of multi-protein networks called 'receptosomes'. These GPCR interacting proteins (GIPs) in the receptosomes control the targeting, trafficking and signaling of GPCRs. PDZ domain proteins constitute the largest protein family among the GIPs, and the predominant function of the PDZ domain proteins is to assemble signaling pathway components into close proximity by recognition of the last four C-terminal amino acids of GPCRs. We present here a machine learning based approach for the identification of GPCR-binding PDZ domain proteins. In order to characterize the network of interactions between amino acid residues that contribute to the stability of the PDZ domain-ligand complex and to encode the complex into a feature vector, amino acid contact matrices and physicochemical distance matrix were constructed and adopted. This novel machine learning based method displayed high performance for the identification of PDZ domain-ligand interactions and allowed the identification of novel GPCR-PDZ domain protein interactions.

• Genomics & Informatics
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• 제20권1호
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• pp.6.1-6.15
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• 2022

Litorilituus sediminis is a Gram-negative, aerobic, novel bacterium under the family of Colwelliaceae, has a stunning hypothetical protein containing domain called von Hippel-Lindau that has significant tumor suppressor activity. Therefore, this study was designed to elucidate the structure and function of the biologically important hypothetical protein EMK97_00595 (QBG34344.1) using several bioinformatics tools. The functional annotation exposed that the hypothetical protein is an extracellular secretory soluble signal peptide and contains the von Hippel-Lindau (VHL; VHL beta) domain that has a significant role in tumor suppression. This domain is conserved throughout evolution, as its homologs are available in various types of the organism like mammals, insects, and nematode. The gene product of VHL has a critical regulatory activity in the ubiquitous oxygen-sensing pathway. This domain has a significant role in inhibiting cell proliferation, angiogenesis progression, kidney cancer, breast cancer, and colon cancer. At last, the current study depicts that the annotated hypothetical protein is linked with tumor suppressor activity which might be of great interest to future research in the higher organism.