• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.676-684
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• 1998

Baculovirus transfer and expression vectors with Hyphantria cunea nuclear polyhedrosis virus (HcNPV) were constructed. An initial transfer vector, pHcEV, constructed using HcNPV was previously reported (Park et al. 1993. J. Kor. Soc. Viral. 23: 141-151). Herein, the size of the vector was properly reduced, and a functionally perfect vector was constructed and named pHcEV-IV (6.7 kb). The vector has a 2.2-kb HcNPV DNA sequence in the 5'-flanking region of the vector's polyhedrin gene promoter. The 1.8-kb HcNPV DNA sequence, poly A signal sequence, T3 primer sequence, and 13 multicloning site sequences, in order, were ligated in front of the translation start codon of the polyhedrin gene. The cloning indicating marker lacZ gene was inserted into the pHcEV-IV, named pHcEV-IV-lacZ, and transferred into the wild-type virus. Recombinant expression virus, lacZ-HcNPV, was constructed by replacing the lacZ gene in the pHcEV-IV-lacZ with the polyhedrin gene of the wild-type virus. The recombinant virus was isolated from blue plaques that produce $\beta$-galactosidase without polyhedra. The lacZ gene insertion was confirmed by Southern hybridization analysis. The expression of the lacZ gene in Spodoptera frugiperda cells infected with the lacZ-HcNPV was examined by SDS-PAGE and colorimetric assay. One 116-kDa LacZ protein band appeared on the PAGE. The production rate of the $\beta$-galactosidase was approximately 50 international units (IU) per min per ml between 2 to 5 days postinfection (p.i.). The highest activity occurred at five days p.i. was 170 IU/min/$m\ell$. The enzyme activity first appeared about 20 h p.i. as measured by colorimetric assay.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.238-243
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• 2000

An exoinulinase (${\beta}-D-fructofuranosidase$) gene was cloned by chromosome walking along the upstream region of the endoinulinase gene of Pseudomonas mucidolens isolated from soil. the exoinulinase gene consisted of an ORF of 0,506 bp encoding a polypeptide of 501 amino acids with a deduced molecular weight of 55,000. The exoinulinase produced by the recombinant Escherichia coli $DH5{\alpha}$ strain was also purified to homogeneity as determined by SDS-PAGE and a zymogram. The molecular weight of the purified exoinulinase according to both SDS-PAGE and gel filtration matched the deduced molecular weight of the protein described above, thereby indicating that the native form of the exoinulinase was a monomer. The purified enzyme hydrolyzed activity value of 2.0. Furthermore, no inulo-oligomers were liberated from the inulin substrate in the enzymatic reaction mixtures incubated for 90 min at $55^{\circ}C$. Taken together, these results indicate that the purified ${\beta}-D-fructofuranosidase$ was an exoinulinase. The pH and temperature optima of the exoinulinase were pH 6.0 and $55^{\circ}C$, respectively. the enzymehad no apparent requirement for a cofactor, and its activity was completely inactivated by $Ag^{+},{\;}Hg^{2+},{\;}and{\;}Zn^{2+}$. Kinetic experiments gave $K_m,{\;}V_{max},{\;}and{\;}K_{cat}$ values for inulin of 11.5 mM, 18 nM/s, and $72{\;}s^{-1}$, respectively. the exoinulinase was fairly stable in broad pH conditions (pH 5-9), and at pH 6.0 it showed a residual activity of about 70% after 4 h incubation at $55^{\circ}C$.

• Journal of Life Science
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• v.10 no.4
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• pp.397-402
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• 2000

Polyphosphate kinase catalyzes the formation of polyphosphate from ATP. To understand the mechanism of phosphate accumulation, the Serratia marcescens gene encoding ppk was cloned from the genomic library by the method of Southern hybridization. The hybridization positive DNA fragment region from pDH3 was subcloned into the expression vector. The ppk gene product, a polypeptide of 75 kDa, was confirmed by SDS-PAGE. Expression of the Serratia marcescens ppk is regulated by the catabolite repression system. The enzyme activity polyphosphate kinase was increased in the E. coli strain harboring plasmid pMH4 with ppk gene.

• Journal of Information Management
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• v.38 no.1
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• pp.21-36
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• 2007

This study aimed to investigate present status of online reference shelf and analyze access point in the home page and types of online reference sources in the university library. Data were collected by accessing home pages of 41 university libraries in the region of Seoul and Kyonggi-Do. 30 libraries had prepared reference shelf out of 41 libraries, and "User Service", "Electronic Reference Sources", or "Reference & Information Sources" were mostly used as home page menu. Types of references sources varied among libraries, and "Directories", "Dictionaries", "Statistics" or "Encyclopedias" were mostly loaded types in the reference shelf. For the enhancement of reference services through reference shelf, establishing and applying standards for evaluation, ease of access, access by subject, and continuous updates and additions of reference sources were suggested.

• Journal of Microbiology
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• v.44 no.1
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• pp.64-71
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• 2006

To investigate the effects of the nucleotide sequences in Shine-Dalgarno (SD) and the spacer region (SD-ATG) on bovine growth hormone (bGH) gene expression, the expression vectors under the control of the T7 promoter (pT7-7 vector) were constructed using bGH derivatives (bGH1 & bGH14) which have different 5'-coding regions and were induced in E. coli BL21 (DE3). Oligonucleotides containing random SD sequences and a spacer region were chemically synthesized and the distance between the SD region and the initiation codon were fixed to nine bases in length. The oligonucleotides were annealed and fused to the bGH1 and bGH14 cDNA, respectively. When the bGH gene was induced with IPTG in E. coli BL21(DE3), some clones containing only bGH14 cDNA produced considerable levels of bGH in the range of $6.9\%\;to\;8.5\%$ of total cell proteins by SDS-PAGE and Western blot. Otherwise, the bGH was not detected in any clones with bGH1 cDNA. Accordingly, the nucleotide sequences of SD and the spacer region affect on bGH expression indicates that the sequences sufficiently destabilize the mRNA secondary structure of the bGH14 gene. When the free energy was calculated from the transcription initiation site to the +51 nucleotide of bGH cDNA using a program of nucleic acid folding and hybridization prediction, the constructs with values below -26.3 kcal/mole (toward minus direction) were not expressed. The constructs with the original sequence of bGH cDNA also did not show any expression, regardless of the free energy values. Thus, the disruption of the mRNA secondary structure may be a major factor regulating bGH expression in the translation initiation process. Accordingly, the first stem-loop among two secondary structures present in the 5'-end region of the bGH gene should be disrupted for the effective expression of bGH.

• 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.

• Journal of Applied Biological Chemistry
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• v.60 no.3
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• pp.257-263
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• 2017

A cellulolytic strain Y2 was isolated from soil obtained in the Canadian Alpine region. The isolate was identified as Paenibacillus sp. Y2 by 16S rRNA sequencing. When grown in LB medium supplemented with carboxymethyl-cellulose (CMC), CMCase production increased to 122.0% of that observed in LB without CMC. Culture supernatant was concentrated by ultrafiltration and 80% ammonium sulfate precipitates were separated by Hi-Trap Q and CHT-II chromatography. The purified enzyme (EG-PY2) showed a homogeneous single band and the molecular mass was estimated to be 38 kDa by SDS-PAGE. Optimum pH and temperature of the enzyme were 4.5 and $30^{\circ}C$, respectively. The half-life of enzyme activity at 50 was 140.7 min, but the enzyme was drastically inactivated within 5 min at $55^{\circ}C$. The enzyme was highly activated to 135.7 and 126.7% by 5.0 mM of $Cu^{2+}$ or $Mg^{2+}$ ions, respectively, and moderately activated by $Ba^{2+}$ and $Ca^{2+}$ ions, whereas it was inhibited to 76.8% by $Fe^{2+}$, and to ${\leq}50%$ by $Mn^{2+}$, $Co^{2+}$, $Zn^{2+}$, and EDTA. The enzyme was activated to 211.5% in the presence of 0.5 M of NaCl and greatly tolerant to 3.15M of NaCl. The enzyme showed 2.98 times higher ${\beta}$-glucanase activity than CMCase activity. Based on these results, it can be concluded that EG-PY2 is an acidophilic, cold-active, and halotolerant endoglucanase. The authors suggest it is considered to be useful for various industrial applications, such as, fruit juice clarification, acidic deinking processes, high-salt food processing, textile and pulp industries, and for biofuel production from seaweeds.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.497-506
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• 1998

The expression in Escherichia coli of a cloned insecticidal protein (ICP) gene from Bacillus thuringiensis var. kurstaki HD1 in pHLN1-80 (+) and pHLN2-80(-) plasmids was investigated through deletions in promoters, transcription start point, and termination region. Six recombinant plasmids were constructed in an attempt to analyze the overexpression of the ICP in relations to its gene structure. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone was not overexpressed which having only -80 bp (contained BtI promoter) part of the ICP gene promoter (without Plac promoter), the right-oriented ICP gene and the termination region. Removal of 350 bp from upstream region of the Plac of the clone pHLN2-80 (-) resulted in overexpression of the ICP. One clone was not overexpressed in which the clone consisted of -72 bp part of the ICP promoter without the transcription start point and the transcriptional termination region, and having the right-oriented ICP gene sequence. One clone consisting of the inverted ICP gene sequence, the -72 bp ICP gene promoter, and without the termination region caused overexpression. One clone which consisted of the inverted ICP gene, the -72 bp ICP gene promoter and the termination sequence was overexpressed. These results indicated that the Plac promoter, transcription termination region, the inverted ICP gene insertion, and the -80 bp or -72 bp part of the ICP gene promoters were concerned in the overexpression of the ICP gene in the recombinant plasmid, and also the overexpression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.

• Journal of Life Science
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• v.20 no.5
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• pp.683-690
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• 2010

The yeast strains of Saccharomyces diastaticus produce one of three isozymes of an extracellular glucoamylase I, II or III, a type of exo-enzyme which can hydrolyse starch to generate glucose molecules from non-reducing ends. These enzymes are encoded by the STA1, STA2 and STA3 genes. Another gene, sporulation-specific glucoamylase (SGA), also exists in the genus Saccharomyces which is very homologous to the STA genes. The SGA has been known to be produced in the cytosol during sporulation. However, we hypothesized that the SGA is capable of being secreted to the extracellular region because of about 20 hydrophobic amino acid residues at the N-terminus which can function as a signal peptide. We expressed the cloned SGA gene in S. diastaticus YIY345. In order to compare the biochemical properties of the extracellular glucoamylase and the SGA, the SGA was purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 chromatography. The molecular weight of the intact SGA was estimated to be about 130 kDa by gel filtration chromatography with high performance liquid chromatography (HPLC) column. Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed it was composed of two heterogeneous subunits, 63 kDa and 68 kDa. The deglycosylation of the SGA generated a new 59 kDa band on the SDS-PAGE analysis, indicating that two subunits are glycosylated but the extent of glycosylation is different between them. The optimum pH and temperature of the SGA were 5.5 and $45^{\circ}C$, respectively, whereas those for the extracellular glucoamylase were 5.0 and $50^{\circ}C$. The SGA were more sensitive to heat and SDS than the extracellular glucoamylase.

• KSBB Journal
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• v.16 no.2
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• pp.188-199
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• 2001

A gene encoding the dextransucrase(dsCB) that synthesizes mostly $\alpha-(1\rightarrow6)$ linked dextran with low amount(10%) of $\alpha-(1\rightarrow3)$ branching was cloned and sequenced from Leuconostoc mesenteroides B-742CB. The 6.1 kbp DNA fragment carrying dsCB showed one open reading frame(ORF) composed of 4,536bp. The deduced amino acid sequence shows that it begins from the start codon(ATG) at position 698 of the cloned DNA fragment and extends to the termination condon(TAA) at position 5,223. The enzyme is consisted of 1,508 amino acids and has an calculated molecular mass of 168.6kDa. This calculated Mw was in good agreement with an activity band of 170kDa on non-denaturing SDS-PAGE. A recombinant E. coli DH5 $alpha$ harboring pDSCB produced extracellular dextransucrase in 2% sucrose medium, and synthesized both soluble and insoluble dextran. To compare the properties of enzyme with B-742CB dextransucrase, the acceptor reaction, hydrolysis of dextran and methylation were performed. The expressed enzyme showed the same properties as B-742CB dextransucrease, but its ability to synthesize $\alpha-(1\rightarrow3)$ branching was lower than that of B-742CB dextransucrase. In order to identify the critical amino acid residues known as conserved regions related to catalytic activity, Asp-492 was replaced with Asn. D492N resulted in a 1.6 fold decrease in specific activity.