• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• The Journal of Natural Sciences
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• v.5 no.1
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• pp.37-45
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• 1992

The gene coding for $\beta$-galactosidase of Neisseria lactamica 2118 was cloned into Escherichia coli MC 1061. The isolated 6.5 Kb EcoR I fragement and 7.2 Kb BamH I fragment of chromosomal DNA in Southern hybridization were ligated to a vector plasmid pBR322 and then transformed into Escherichia coli MC 1061 cells. Finally, I obtained three clones as $\beta$-galactosidase positive clone by colony hybridization and Southern hybridization($\beta$-galactosidase probe: lac Z gene of pMC1871). Three recombinant plasmids(pNL.13. 17 and 24) were found to contain the 7.2Kb BamH I fragment originated from Neisseria lactamica 2118 chromosomal DNA by Southern hybridization and pNL 24 was showed high homology to probe especially and also its physical map was constructed.

• The Korean Journal of Zoology
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• v.38 no.4
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• pp.550-556
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• 1995

The c-myc proto-oncogene is Involved In the control of normal cell proliferation and differentiation of many cell lineages. Although it has heen suggested that c-myc may play an important role in the mammalian early development, it Is unclear whether the embryonic c-myc mRNA is originated from zygotic gene expression or stored maternal message. Thus, we have construded expression vectors, In which the 5, flanking sequences including c-myc promoter region and a large non-coding exon I are fused 'sith E. coli lacZ gene that encedes $\beta$-galactosldase as a reporter. As c-myc exon I contains a modulatory sequence, we designed t, vo types of vectors (pcmyc.Gall and pcmyc-Ga12) to examine the role of exon I in c-myc expression. The former contains the complete exon I and the later has a deletion in 40 bp of modulator sequence located In the exon I of c-myc These vectors were microInjected into fertilized one-cell embryos and $\beta$-galactosidase activity was examined by X-gal staining during early embryogenesis. $\beta$-galactosidase activity derived from c-myc promoter was decreased at two-cell stage. The expression level directed by pcmyc- Ga12 was similar to that of pcmyc-Gal1, indicating that the medulatory sequence in exon I may not be Involved at least In the regulation of embryonic c-myc expression. In summary, the present study indicates that the c-myc promoter is functional at the early stage embryo, and the regulation of c-myc expression is under the control of "zygotic" clock of preimplantation mouse embryos.e embryos.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.239-255
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• 1997

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the pMal-pol clone (Lee et al. 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind T5 phage promoter and the $6{\times}His$ region of the pQE-3D expression vector, and it was called pQEVPl. Again, the $6{\times}$His-tagged VP1 DNA fragment in the pQEVP1 was cleaved with EcoRI and transferred into the VP1 site of the pBacVP1, resulting pBacHis-VP1 recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay. The recombinant virus was named VP1-HcNPV-1. The $6{\times}$His-tagged VP1 protein produced by the pQEVP1 was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was $2.0{\times}10^5\;pfu/ml$ at 7 days postinfection.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.5
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• pp.498-502
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• 1998

The present study was designed, fIrst to develop the new methodology to measure the bioluminescence activity easily in live developing bovine embryos by photoncounting, and secondly to compare the expression efficiency of four luciferase reporter genes in bovine embryos at four- to 16-cell stages. In experiment 1, equimolar pSVlacZ and pSVEluc were microinjected into the pronucleus of fertilized bovine oocytes. At 2 days after micro injection, bioluminescence activity of these embryos was measured by photoncounting with a luminometer for 1 min, and lacZ gene expression in the same embryos was assayed by X-gal staining. All the luciferase-positive oocytes showed some bacterial ${\beta}$-galactosidase activity irrespective of the intensity. In experiment 2, four firefly luciferase genes (pTKEluc, pTK6WEluc, pSVEluc and pMiwluc) were introduced by micro injection, and the injected embryos were cultured for the following 2 days. Detection of the luciferase gene expression was done by photoncounting at 5 to 55 min. Over the measurement period, the luciferase activity was almost constant irrespective of the transgenes microinjected. The luciferase activity and expression efficiency at 2 days after microinjection were not significantly affected by the difference in the microinjected transgenes. The present results demonstrated that the bioluminescence activity in live developing bovine embryos could be measured quickly by photoncounting.

• BMB Reports
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• v.43 no.7
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• pp.468-473
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• 2010

Previously, various inhibitors of cell wall synthesis induced the drp35 gene of Staphylococcus aureus efficiently. To determine whether drp35 could be exploited in antistaphylococcal drug discovery, we cloned the promoter of drp35 ($P_d$) and developed different biological assay systems using an engineered S. aureus strain that harbors a chromosomally-integrated $P_d$ - lacZ transcriptional fusion. An agarose-based assay showed that $P_d$ is induced not only by the cell wall-affecting antibiotics but also by rifampicin and ciprofloxacin. In contrast, a liquid medium-based assay revealed the induction of $P_d$ specifically by the cell wall-affecting antibiotics. Induction of $P_d$ by sublethal concentrations of cell wall-affecting antibiotics was even assessable in a microtiter plate assay format, indicating that this assay system could be potentially used for high-throughput screening of new cell wall-inhibiting compounds.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.5
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• pp.320-326
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• 2000

Promoters of the genes G3P, ICL1, POT1, POX1, POX2 and POX5 of the yeast Y. lipolytica were studied in respect to their regulations and activities during growth on different carbon sources. The aim of this study was to select suitable promoters for high expression of heterologous genes in this yeast. For this purpose the promoters were fused with the reporter gene lacZ of E. coli and integrated as single copies into the genome of Y. lipolytica strain PO1d. The measurement of expressed activities of ${\beta}$-galactosidase revealed that pICL1, pPOX2 and pPOT1 are the strongest regulable promoters available for Y. lipolytica, at present. pPOX2 and pPOT1 were highly induced during growth on oleic acid and were completely repressed by glucose and glycerol. pICL1 was strongly inducible by ethanol besides alkanes and fatty acids, however, not completely repressible by glucose or glycerol. Ricinoleic acid methyl ester appeared as a very strong inducer for pPOT1 and pPOX2, in spite of that it inhibited growth of Y. lipolytica transformants.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.336-339
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• 2002

A total of 100 Corynebacterial clones exerting a regulatory effect on the aceB promoter of Corynebacterium glutamicum were isolated by utilizing a reporter carrying the enteric lacZ gene fused to the promoter. The isolated clones were classified into 3 groups of A, B, and C, according to their color of colonies. Escherichia coli cells carrying clones in groups A and B showed a $90\%\;and\;50\%$ reduction in ${\beta}$-galactosidase activity, respectively. The introduction of group A clones into C. glutamicum also resulted in an almost complete reduction in the expression of the aceA and aceB genes, suggesting that the clones express repressor-like proteins for the genes. Although white colonies were formed on plates containing X-gal, E. coli cells carrying one of the clones in group C exhibited intact ${\beta}$-galactosidase activity. The result suggests that the clone may encode proteins that prevent the cells from accumulating the chromogenic compound, X-gal.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.226-233
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• 2007

Members of Methylobacterium, referred as pink-pigmented facultative methylotrophic bacteria, are frequently associated with terrestrial and aquatic plants, tending to form aggregates on the phyllosphere. We report here that the production of autoinducer molecules involved in the cell-to-cell signaling process, which is known as quorum sensing, is common among Methylobacterium species. Several strains of Methylobacterium were tested for their ability to produce N-acyl-homoserine lactone (AHL) signal molecules using different indicators. Most strains of Methylobacterium tested could elicit a positive response in Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. The synthesis of these compounds was cell-density dependent, and the maximal activity was reached during the late exponential to stationary phases. The bacterial extracts were separated by thin-layer chromatography and bioassayed with A. tumefaciens NTI (traR, tra::lacZ749). They revealed the production of various patterns of the signal molecules, which are strain dependent. At least two signal molecules could be detected in most of the strains tested, and comparison of their relative mobilities suggested that they are homologs of N-octanoyl-$_{DL}$-homoserine lactone ($C_8-HSL$) and N-decanoyl-$_{DL}$-homoserine lactone ($C_{10}-HSL$).

• Korean Journal of Microbiology
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• v.45 no.2
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• pp.215-221
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• 2009

Site-specific incorporation of unnatural amino acids into proteins in vivo can be achieved by co-expression of an orthogonal pair of suppressor tRNA and engineered aminoacyl-tRNA synthetase (ARS) that specifically ligates an unnatural amino acid to the suppressor tRNA. As a step to establish this technique, here we generated an Escherichia coli reporter strain DH10B(Tn:lacZam) by integrating amber mutated lacZ gene into the chromosome of E. coli DH10B strain. In vivo expression of E. coli amber suppressor $tRNA^{Gln}$ produced blue colonies in culture plates containing X-Gal as well as dramatically increased $\beta$-galactosidase activity. In addition, expression of an orthogonal pair of Saccharomyces cerevisiae suppressor $tRNA^{Tyr}$ and tyrosyl-tRNA synthetase also produced blue colonies as well as moderate increase of $\beta$-galactosidase activity. These data demonstrate that our reporter strain will provide an efficient method to assess amber suppression in both qualitative and quantitative manners.