• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.301-305
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• 2002

The gene (ppaT) encoding Thermus caldophilus GK24 pyrophosphatase (Tca pyrophosphatase) was cloned and sequenced. The gene was found to contain an open reading frame encoding 175 amino acids with a calculated mass of 19,155 Da. The ppaT gene was expressed under the control of the tac promoter in Escherichia coli. The recombinant Tca pyrophosphatase was purified 21.4-fold with $56\%$ yield and specific activity of 25.7 U $mg^-1$, following a combination of heating (to denature the E. coli proteins) and one step of DEAE-Sephacel column chromatography. The native enzyme was found to have an approximate molecular mass of 110,000 Da and consisted of six subunits. The enzyme exhibited maximal activity at pH of 8.0-8.5 and was stable at $80-90^{\circ}C$. A divalent cation was absolutely required for the enzyme activity, with $Mg^2+$. being the most effective.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6803-6806
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• 2013

Background: Interferon regulatory factor 6 (IRF6) is a transcription factor with distinct and conserved DNA and protein binding domains. Mutations within the protein binding domain have been significantly observed in subjects with orofacial cleft relative to healthy controls. In addition, recent studies have identified loss of expression of IRF6 due to promoter hypermethylation in cutaneous squamous cell carcinomas. Since mutational events occurring within the conserved domains are likely to affect the function of a protein, we investigated whether regions within the IRF6 gene that encodes for the conserved protein binding domain carried mutations in oral squamous cell carcinoma (OSCC). Materials and Methods: Total chromosomal DNA extracted from 32 post surgical OSCC tissue samples were amplified using intronic primers flanking the exon 7 of IRF6 gene, which encodes for the major region of protein binding domain. The PCR amplicons from all the samples were subsequently resolved in a 1.2% agarose gel, purified and subjected to direct sequencing to screen for mutations. Results: Sequencing analysis resulted in the identification of a mutation within exon 7 of IRF6 that occurred in heterozygous condition in 9% (3/32) of OSCC samples. The wild type codon TTC at position 252 coding for phenylalanine was found to be mutated to TAC that coded for tyrosine (F252Y). Conclusions: The present study identified for the first time a novel mutation within the conserved protein binding domain of IRF6 gene in tissue samples of subjects with OSCC.

• 대한바이러스학회지
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• 제27권2호
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• pp.151-160
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• 1997

In view of the potential of replicase protein as a diagnostic reagent for human caliciviruses (HuCVs), we have cloned and over-expressed this gene from the Snow Mountain-like Korean strains in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and described the preliminary antigenic characterization of the recombinant products. Each 470bp fragment corresponding to highly conserved region of RNA-dependent RNA polymerase was generated by RT-PCR from stools of two diarrheal children, cloned in pMOSBlue T-vector, and subcloned between the EcoRI and SalI restriction sites of pGEX-4T-3, a GST gene fusion vector, yielding $pGCV_{pol}$. This construct expressed a Snow Mountain-like HuCV replicase under the control of the IPTG-inducible tac promoter. An extract prepared by sonication of the E. coli cell inclusion bodies bearing $pGCV_{pol}$ products was purified and analyzed by SDS-PAGE. After Coomassie blue staining, it was shown that the recombinant replicase migrated on the gels with an approximate molecular mass of 46.5 kDa, that was subsequently cleaved into a 26 kDa GST fragment and a 20.5 kDa replicase protein upon digestion with thrombin protease. The replicase was recognized on immunoblotting with the sera from symptomatic children with the HuCV-associated diarrhea but not by asymptomatic sera from adults. The results presented the first biological activity of individually expressed HuCV replicase subunit and provided important reagents for diagnosis of HuCV infection.

• BMB Reports
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• 제34권4호
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• pp.365-370
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• 2001

We constructed a comparative expression system in order to produce recombinant human ferritin homo- and heteropolymers in Escherichia coli. Human ferritin H-(hfH) and L-chain (hfL) genes were expressed without amino acid changes under the control of a tac promoter. Ferritin heteropolymers of varying subunit composition were also produced by combining two different expression systems, a bicistronic expression system and a coplasmid expression system. As a result, recombinant H-chain ferritin and ferritin heteropolymers were catalytically active in forming iron core in vivo. In particular, the ferritin heteropolymer that is composed of 7% H-subunit and 93% L-subunit was capable of forming an iron core of the protein, while the L-chain ferritin homopolymer was inactive in vivo. This result indicates that the two H-subunits (i.e., 7% H-subunit content) are important to keep ferritin active in the cells. In addition, human ferritins were identified as the major iron binding proteins in the transformed cells. Also, the amount of iron bound to the recombinant ferritins was proportional to the H-subunit content in ferritin heteropolymers in vivo.