• Korean Journal of Veterinary Research
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• v.45 no.2
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• pp.185-189
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• 2005

Actinobacillus pleuropneumoniae is the causative agent of a porcine contagious pleuropneumonia. Among several virulence factors including exotoxin (Apx toxins), LPS, transferrin-binding proteins, OMPs, and some proteases, Apx toxins have been major targets for the protection study. In this study, cloning and expression of A. pleuropneumoniae Apx I and Apx II toxin, which are produced by all highly virulent strains, were performed by Escherichia coli expression system. Genes coding Apx I and II toxin were amplified from the A. pleuropneumoniae serotype 5 genomic DNA using polymerase chain reaction and cloned to a prokaryotic expression vector, pRSET. Expression of the Apx I and Apx II coding sequences in E. coli resulted in the formation of insoluble inclusion bodies purified according to a denaturing purification protocol, which employs the use of guanidium. Recombinant proteins were purified using $Ni^{2+}$-charged resin affinity purification. This expression and purification system made it possible to produce Apx I and Apx II in large amounts for further immunologic studies.

• Journal of the Korean Magnetic Resonance Society
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• v.14 no.2
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• pp.105-116
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• 2010

SH3YL1, a novel protein containing one Src homology 3 domain at the carboxyl terminus was first detected in mouse anagen skin cDNA. This protein had a significant homology with YHRO 16c/Ysc 84, the yeast Src homology 3 domain-containing protein. The sequence identity was remarkable at the carboxyl and amino-terminal Src homology 3 domain, suggesting that the novel protein is a mouse homolog of the yeast protein and thus was termed as SH3YL1. SH3YL1 is composed of two domains, a DUF500 at N-termini and a SH3 domain at C-termini. In our study we cloned the SH3 domain in bacterial expression system in Escherichia coli using pET32a vector with TEV protease cleavage site and purified as a monomer using affinity chromatography. The N-terminal poly-Histidine tag was cleaved with TEV protease and target protein was used for backbone studies. Our study showed that SH3 domain primarily consists of $\beta$-sheet which is in consistence with previous result performed on the truncated SH3 domain of SH3YL1.

• Journal of the Korean Magnetic Resonance Society
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• v.16 no.2
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• pp.147-161
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• 2012

Human melanocortin-4 receptor (hMC4R) among MC-Rs, expressed in the brain, is in charge of the control on energy homeostasis and food intake. The structure and function of human MC4R have been studied to understand their essential function and roles. To investigate the structure and function, it is necessary to prepare sufficient amounts of proteins. However, their expression and purification is demanding and time-consuming due to their innate insoluble and toxic properties. The heterozygous mutations of hMC4R, exchange of Asp 90 to Asn located in second transmembrane, cause severe obesity in human. To obtain purified hMC4R wt-TM2 for structural studies, it was first over-expressed and purified by fast protein liquid chromatography (FPLC) and then solution NMR studies were performed to get high-resolution spectra. In here, we established optimized purification scheme to get more purified target peptide.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.274-279
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• 2015

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-_D-1-thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.887-892
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• 2015

Uricase is an important microbial enzyme that can be used in the clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of the uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuDof P. aeruginosa PAO1. We report the successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about 58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. We also established efficient protein purification using the Ni-Sepharose column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific activity of the pure enzyme compared with the crude enzyme.

• Korean Journal of Plant Tissue Culture
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• v.27 no.4
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• pp.283-297
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• 2000

Seeds are an ideal repository for recombinant proteins in molecular farming applications. However, in order to use plant seeds efficiently for the production of such proteins, it is necessary to understand a number of fundamental biological properties of seeds. This includes a full understanding of promoters which function in a seed-specific manner, the subcellular targeting of the desired polypeptide and the final form in which a protein is stored. Once a biologically active protein has been deposited in a seed, it is also critical that the protein can be extracted and purified efficiently. In this review, these issues are examined critically to provide a number of approaches which may be adopted for production of recombinant proteins in plants. Particular attention is paid to the relationship between subcellular localization and protein extraction and purification. The robustness and flexibility of seed-based production is illustrated by examples close to or already in commercial production.

• Biomedical Science Letters
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• v.2 no.2
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• pp.231-240
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• 1996

Human blood clotting (coagulation) factor 9 cDNA which codes for 461 amino acid has been cloned by screening human fetal liver cDNA library using PCR. This 1.4 kb cDNA spanning from the ATG initiation codon to the TAA termination codon was cloned into bacterial .expression vector pGEX-2T, generating pGEX-F9 plasmid. The plasmid pGEX-F9 expresses about 73 kDa GST (Glutathione S-transferase)-Factor 9 fusion protein when introduced into E. coli. Western blot analysis using polyclonal antibody raised against human factor 9 confirmed this fusion protein contains factor 9 protein. The level of GST-factor 9 expression was about 20% of total protein and the purification of fusion protein was efficiently achieved by using GST agarose bead based on one step purification protocol.

• KSBB Journal
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• v.13 no.6
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• pp.656-661
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• 1998

Hantaan virus belonging to the genus Hantavirus and family Bunyaviridae causes an acute severe illness of human, Haemorrhagic Fever with Renal Syndrome (HFRS). It is a rodent host-borne pathogen and distributed in Asia and Eastern Europe. Hantaviruses have three major antigens, i.e., G1, G2 glycoproteins and nucleocapsid protein (N). Among them, nucleocapsid protein was reported to be the most invaluable antigen as for diagnosis. We have cloned and expressed Hantaan viral nucleocapsid gene in E. coli BL21(DE3). In this study, we have tried to purify the nucleocapsid protein produced by recombinant E. coli, and could attained a purity of >90% by anti-N monoclonal antibody-coupled immunoaffinity chromatography or phenyl sepharose hydrophobic interaction chromatography.

• Journal of Life Science
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• v.21 no.8
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• pp.1067-1075
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• 2011

Coiled-coil domain-containing protein 98 (CCDC98) plays a role in G2/M DNA damage checkpoint pathways by recruiting breast cancer 1 (BRCA1)-A complex to the DNA-damaged sites. However, the molecular mechanism of CCDC98 on the DNA damage-induced G2/M checkpoint pathways is unclear. In this study, we identifed Wilms tumor 1-associating protein (WTAP) as a novel CCDC98-binding protein, using tandem affinity purification. We confirmed the association between CCDC98 and WTAP using in vivo and in vitro binding assays. We demonstrated that CCDC98 regulates cyclin B1 expression by affecting WTAP protein stability. Based on these results, we suggest that CCDC98 may act as a novel cell cycle regulator by regulating the expression level of cyclin B1.

• KSBB Journal
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• v.30 no.6
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• pp.291-295
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• 2015

The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with $CaCl_2$ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.