• Microbiology and Biotechnology Letters
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• v.28 no.1
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• pp.21-25
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• 2000

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in Escherichia. coli TP2139 ( lac, crp). One of the cloned genes, pCKB17, was further analyzed. In order to find whether the increased expression of the gent was under the direction of maltose metabolism, we constructed several recombinant subclones. We have found that the clone, pCKB17AV, codes maltose metabolism stimulation(mms) gene. E. coli transformed with the cloned gene showed increase in the activity of maltose utilzation, The recombinant proteins expressed by multicopy and induction with IPTG, one polypeptide of 29-kDa, was confirmed by SDS-PAGE. The overexpression of maltose-binding proter protein in the presence of mms gene was confirmed by Western blot analysis. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.145-149
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• 1995

For the purpose of developing a new biodegradable detergent, we have isolated a gene encoding wide-range temperature applicable alkaline protease from Xanthomonas sp. YL-37 (Lee et al., 1994, Kor. J. Appl. Microbiol. Biotechnol.). An alkaline protease gene was isolated from the gene bank that was prepared from the chromosomal DNA of Xanthomonas sp. YL-37. From the results of agarose gel electrophoresis and a restriction enzyme mapping, a 2.7 kb DNA fragment containing the alkaline protease gene was inserted in the plasmid pUC9. Extracellular activity of a clone having alkaline protease gene was detected on SDS-polyacrylamide gel with activity staining assay. The molecular weight of alkaline protease was determined to be about 64 kDa from 11% SDS-PAGE analysis. Alkaline protease activity, produced from E. coli which harboring the plasmid, showed no difference at reaction temperature 20, 30 and 40$\circ$C, respectively. This result showed that alkaline protease produced from E. coli harboring the plasmid was apparently the same as that of Xanthomonas sp. YL-37.

• Journal of Microbiology
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• v.44 no.1
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• pp.54-63
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• 2006

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the $^{32}P-labeled$ partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5' -untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.

• Biomolecules & Therapeutics
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• v.8 no.2
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• pp.153-159
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• 2000

A novel serine/threonine protein phosphatase with EF-hand motif, which belongs to PPEF family was partially cloned from rat brain cDNA by employing RT-PCR method. The size of the amplified clone was 1.6kbp. The amplified DNA was subcloned into pGEM-T-Easy vector and the resulting plasmid was maned as pGEM-rPPEF2. The nucleuotide sequence is shared by 88% with that of mouse PPEF-2 cDNA, and the deduced amino acid sequence reveal 92% homology with that of mouse PPEF-2 cDNA. The N-terminal region of the cloned rat brain PPEF contains a putative phosphatase catalytic domain (PP domain) and the C-terminal region contains multiple $Ca^{2+}$ binding sites (EF region). The putative catalytic domin (PP) and the EF-hand motif (EF) regions were subcloned into pGEX4T-1 and were overexpressed in E. coli DH5 as glutathione-S-transferase (GST) fusion proteins. Expression of the desired fusion protein was identified by SDS-PAGE and also by immunoblot analysis using monoclonal antibody against GST. The recombinant proteins were purified by glutathione-agarose chromatography. This report is first to demonstrate the cloning of PPEF family from rat brain tissues. The clone reported here would be invaluable for the investigation of the role of this new type-phosphatase in rat brain.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.3
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• pp.480-487
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• 1997

Vitellogenin (Vg) was purified from plasma of $estradiol-17\beta(E_2)-treated$ spotted flounder, verasper variegatus, by preripitation with cold distilled water, followed by fractionation using a Sepharose CL-6B column chromatography. $E_2-induced$ protein was identified as Vg by SDS-PAGE and western blot analysis. The molecular weight of the purified Vg was estimated 175 kD as determined by SDS-PAGE. In order to measure the Vg level, monoclonal antibodies against Vg were produced by hybridoma technique. Purified Vg was immunized into Balb/c mice and then the spleen cells from mice were fused with NS-1 myeloma cells. The hybridoma cells were screened by enzyme-linked immunosorbent assay (WLISA) and subcloned by limiting dilution. The hybridoma clones which secreted antibodies highly reactive to the purified Vg were designated as 4D6. Its specificity was demonstrated by western blot from plasma of untreated, $E_2-treated$ male fish, and purified Vg.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.4
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• pp.561-566
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• 2014

The complete coding sequence of Wild Argali ISG15 cDNA was generated by rapid amplification of cDNA ends. The ISG15 cDNA was 642 bp with an open reading frame of 474 bp, which encoded a 17.47 kDa protein composed of 157 amino acids. Its amino acid sequence shared 97.9%, 80.8%, 91.4%, 94.3%, 78.3% identity with those of ISG15cDNA from Ovis aries (accession no. NM001009735.1), Capra hircus (accession no. HQ329186.1), Bos taurus (accession no. BC102318.1), Bubalus bubalis (accession no. HM543269.1), and Sus scrofa (accession no. EU647216.1), respectively. The entire coding sequence was inserted into the pET-28a vector and expressed in E. coli. The recombinant protein corresponded to the expected molecular mass of 25 kDa as judged by SDS-PAGE, and it was detected in the bacterial inclusion bodies. The expressed protein could be purified by $Ni^{2+}$ chelate affinity chromatography and the results from the lymphocyte proliferation test showed that the product could stimulate lymphocyte proliferation very well (p<0.05), which further confirmed its biological activity.

• BMB Reports
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• v.43 no.5
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• pp.375-381
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• 2010

In this study, the cDNA library of Chang-liver cells was immunoscreened using common ADAMs antibody to obtain ADAM related genes. We found one positive clone that was confirmed as a new gene by Blast, which is an uncharacterized helical and coil protein and processes protease activity, and named protease-related protein 1 (ARP1). The submitted GenBank accession number is AY078070. Molecular characterizations of ARP1 were analyzed with appropriate bioinformatics software. To analyse its expression and function, ARP1 was subcloned into glutathione S-transferase fusion plasmid pGEX-2T and expressed by E. coli system. The in vitro expression product of ARP1 was recognized by common ADAMs antibody with western blot. Interestingly, ARP1 cleaves gelatine at pH9.5, which suggests it is an alkaline protease. Semi-quantitative RT-PCR result indicates that ARP1 mRNA is strongly transcribed in the liver and the treated Chang-liver cells.

• Journal of Life Science
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• v.9 no.1
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• pp.54-57
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• 1999

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in E. coli TP2139 (${\Delta}$lac, ${\Delta}$crp). One of the cloned genes, pCKB13, was further analyzed. In order to find whether the increased expression of the gene under the direction of maltose metabolism, we constructed several recombinant subclones. We have confirmed that the clone, pCKB13 codes Coenzyme A transferase gene by partial nucleotide sequencing in the terminal region. The enzyme activity of Coenzyme A transferase increased after introduction of the multicopy of the cloned gene in E. coli. The recombinant proteins expressed by multicopy and induction with IPTG, two polypeptide of 26-and 28-kDa, were confirmed by SDS-PAGE. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.494-498
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• 1989

Hybridoma cell lines, which secrete monoclonal antibodies (mAbs) against the surface antigens of Cryptosporidium parvum Sporozoites, were produced by fusing spleen cells of C. parvum Sporozoite-immunized mice with P3-X63-Ag8 myeloma cells. Two cloned antibody-secreting cell lines, Kor1 and Ea2, were established and produced IgG1 and IgG2a antibodies, respectively. Percoll-purified sporozoites were solubilized and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Western blot assay demonstrates that an antigen of 20-kDa was bound by monoclonals. By indirect immunofluorescence microscopy, mAb exhibited uniform binding to the sporozoite surface.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.43-48
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• 1993

These experiments were carried out to construct mouse testis cDNA library and to to seen H-Y Ag gene. Mouse testis was obtained from BALB/c inbreed mouse that was after-born 1 week. Isolation of mouse testis total RNA was carried out by guanidum/cesium choloride, poly(A+) mRNAs were purified by oligo d(T)-cellulose chromatography method. To investigate protein synthesis activity, in-vitro translation carried out by total RNA and poly(A+) mRNA. The products of in-vitro translation were identified in 12.5% PAGE. Single strand DNA and double strand DNA were synthesized from poly(A+) mRNA and purified using phenol/chloroform/isoamylalcohol. Synthesized cDNA was combined with cohesive Eco RI polylinker, its recombination efficiencies were identified by X-gal and IPTG. In the cDNA library, 1$\times$107 phagemids were screened with 32P labelled probe. Hybridization were carried on $65^{\circ}C$ for 16~20hours. And 1$\times$106 phagemids were screened with rabbit-anti-H-Y. In former, select 5 positive clones, and later, 1 positive clone. Its southern blot analysis showed various size of insert cDNA from 0.7kb to 3kb.