• Journal of Microbiology
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• v.34 no.1
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• pp.74-81
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• 1996

Two secretion vectors, pUBA240 and pUB340 were constructed by using the promoter and secretory signal region of the .alpha.-amylase gene from an .alpha.-amylase hyperproducing strain, Bacillus subtilis NA64. In this secretion vector system, various restriction enzyme sites are located immediately after the proregion of the .alpha.-amylase gene for easy replacement of various foregn structural genes. To evaluate this secretion vectors, the .betha.-lactamase gene of pBR322 was used as a reporter gene. The expressed and biologically active .betha.-lactamase was secreted into the culture broth from B. subtilis LKS86 transformants harboring each .betha.-lactamase secreting plasmid, pUBAbla and pUBSble. In both cases, more than 92% of expressed .betha.- lactamases were located idn the culture medium. The amount of the secreted .betha.-lactamase was about 80% of the total secreted proteins in the culture medium.

• Korean Journal of Microbiology
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• v.23 no.2
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• pp.122-128
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• 1985

Bacillus thuringiensis serovar israelensis 4Q1 contains 8 different covalently closed-circular (CCC) plasmids of molecular weight 204, 267, 109, 103, 16, 7.6, 6.4, and 5.0kb. The three smallest plasmids, designated pBti6, and pBti8 may prove to be useful as cloning vectors because of thier size and ease of isolation. The three plasmids were incubated separately with 9 different restriction enzymes and 7 of the enzymes tested cleaved one or more of the plasmids. Plasmid pBti6 has a single site for Bg1 II, Pst I and Pvu II, two sites for Bc1 I and Eco RI, and five sites for Hind III. Plasmid pBti7 has a single site for Bam HI and Pst I, two sites for Hind III, and three sites for PvuII. Plasmic pBti8 has a single site for Bam HI, BelI and Hind III, two sites for Eco RI, and three sites for Bgl II and Pvu II. Composite restriction enzyme maps for pBti6, pBti7 and pBti8 were constructed. The sites of restriction enzyme cleavage were determined by single, double and partial digests of the plasmid DNA. All the restriction sites were aligned relative to the single Bgl II(pBti6), Pst I(pBti7) or Hind III(pBti8) site, respectively.

• Proceedings of the Korean Society of Life Science Conference
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• 2001.11a
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• pp.3-8
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• 2001

Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all carotenoids, IPP in Escherichia coli is synthesized through the non-mevalonate pathway. The first reaction of IPP biosynthesis in E. coli is the formation of 1-deoxy-D-xylulose-5-phosphate (DXP), catalyzed by DXP synthase and encoded by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phosphate, catalyzed by DXP reductoisomerase and encoded by dxr. To determine if one or more of the reactions in the non-mevalonate pathway controlled flux to IPP, dxs and dxr were placed on several expression vectors under the control of three different promoters and transformed into three E. coli strains (DH5(, XL1-Blue, and JM101) that had been engineered to produce lycopene. Lycopene production was improved significantly in strains transformed with the dxs expression vectors. When the dxs gene was expressed from the arabinose-inducible araBAD promoter (PBAD) on a medium-copy plasmid, lycopene production was 2-fold higher than when dxs was expressed from the IPTG-inducible trc and lac promoters (Ptrc and Plac, respectively) on medium-copy and high-copy plasmids, Given the low final densities of cells expressing dxs from IPTG-inducible promoters, the low lycopene production was probably due to the metabolic burden of plasmid maintenance and an excessive drain of central metabolic intermediates. At arabinose concentrations between 0 and 1.33 mM, cells expressing both dxs and dxr from PBAD on a medium-copy plasmid produced 1.4 - 2.0 times more lycopene than cells expressing dxs only. However, at higher arabinose concentrations lycopene production in cells expressing both dxs and dxr was lower than in cells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-inducible dxs on a medium-copy plamid revealed that lycopene production was highest in XL1-Blue.

• Bulletin of the Korean Chemical Society
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• v.33 no.11
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• pp.3676-3680
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• 2012

An Epstein-Barr virus (EBV)-based plasmid contains the EBV nuclear antigen 1 (EBNA1) gene and EBV replication origin (oriP) sequence. Since EBNA1 (the only EBV-encoded protein) is combined with oriP, it is replicated simultaneously with chromosomal DNA in human, primate, and canine cells and is faithfully segregated at a stable copy number upon cell division. Consequently, it can be used to stably express gene inserts over a prolonged time in target cells. We have previously shown that the polyamidoamine (PAMAM) dendrimer can be surface-modified with L-arginine. Arginine is present at a high frequency in the transactivator of transcription (Tat) sequences of human immunodeficiency virus (HIV). It presents high membrane permeability and permits effective transfer of DNA inside the cells. In this study, we constructed two kinds of recombinant DNA by inserting the luciferase gene and enhanced green fluorescence protein (eGFP) gene as reporter genes into the pCEP4 plasmid vector. We measured dynamic light scattering (DLS) and zeta potential after preparing PAMAM-based cationic polymer/EBV-based plasmid complexes. We performed transfection of HEK 293 cell lines with the polyplexes, and monitored luciferase activity and green fluorescence protein (GFP) expression. Our results show that PAMAM-based cationic polymer/EBV plasmid complexes provide enhanced and sustained gene expression.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.156-163
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• 1995

Bacillus sphaericus 1593 is pathogenic to the larvae of a number of mosquito species that are known as important vectors for the transmission of certain human and animal diseases. As a preliminary experiment for developing a multfunctional B. sphaericus 1593 as a potent antagonist, we investigated the conditions for the protoplast transformation system of B. sphaericus 1593 using the plasmid pGB215-110$\Delta$B. The protoplast of B. sphaericus 1593 were obtained most efficiency by treating the cells with 500 $\mu$g/ml of lysozyme in the SMM buffer containing 0.5 M sucrose at pH 8.0 and 40$\circ$C for 60 minutes. The cell wall was regenerated on the plate containing 1.2% agar and 0.8 M mannitol. Under the best condition for protoplast formation and regeneration established in the work the highest frequency of transformation was achieved with the 40% PEG (M.W 4,000) treatment for 15 minutes of incubation at 4$\circ$C, and subsequently for 120 minutes incubation at 30$\circ$C for phenotypic expression. The highest transformation efficiency were observed at 1.0 $\mu$g/ml of the final concentration of the plasmid DNA and the plasmids were found to be fairly stable since about 70% of the plasmids were maintained after 8 successive daily transfers onto the fresh medium.

• Journal of Pharmaceutical Investigation
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• v.40 no.6
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• pp.357-362
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• 2010

To enhance therapeutic effects of insulin-sensitizing adipokine, ADN gene and potent agonists, rosiglitazone for the $PPAR{\gamma}$, cationic liposomes as non-viral vectors were formulated. The particle size and zeta potential of drug loaded and unloaded cationic liposomes were investigated. The complex formation between cationic liposomes and negatively charged plasmid DNA was confirmed and the protection from DNase was observed. In vitro transfection was investigated in HepG2, HeLa, and HEK293 cells by mRNA expression of ADN. Encapsulation efficacy of rosiglitazone-loaded liposomes was determined by UV detection. Particle sizes of cationic liposomes were in the range of 110-170 nm and those of rosiglitazone-loaded cationic liposomes were in the range of 130-180 nm, respectively. Gel retardation of complexes indicated that the complex was formed at weight ratios of cationic lipid to plasmid DNA higher than 20:1. Both complexes protected plasmid DNA from DNase either drug free or drug loading. Encapsulation efficiency of rosiglitazone-loaded emulsion was increased by drug dose. The mRNA expression levels of ADN were dose-dependently increased in cells transfected with plasmid DNA. Therefore, cationic liposomes could be potential co-delivery system for drug and gene.

• KSBB Journal
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• v.13 no.4
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• pp.418-423
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• 1998

Cellular transfections with cationic liposomes are widely empolyed for gene and oligonucleotide transfer in vitro because of their safety and ease of use. However, they still suffer from the low transfection efficiency comparing with viral vectors. Substantial effort shave been focused on increasing transfection efficiency by supplementing the liposome/DNA complexes(lipoplex) with various components. In this work, we tired three kinds of supplements, Poly-L-lysine(PLL), transferrin and a mixture of anionic lipids(PS/PE/PC), to study their effects on gene transfer yield and gene expression efficiency. PLL, a polycationic polymer, enhanced gene transfer yield by 3 times but the gene expression efficiency was increased only by 1.5 times. this result implies that PLL can enhance the transfection efficiency mainly by increasing the rate of outermembrane transport of lipoplex into the cells. On the other hand, transferrin which can facilitate the gene transfer via ligand-receptor interaction gave not only increased gene transfer yield but also enhanced gen expression efficiency by 2.8 times. Transferrin seems to contribute to the escape of plasmid from endosomes through ligand-receptor recycle mechanism. When the cells were treated with a mixture of anionic lipids for 3 hours before the transfection, gene transfer yield was slightly decreased but the gene expression efficiency was enhanced by 1.9 times. This is presumably due to the accelerated liposome-plasmid dissociation by the anionic lipids, and the increased delivery of plasmid to the nucleus. According to these results, it is clear that the supplementation to ameliorate transfection efficiency with cationic liposomes should be contrived in the direction of increasing delivery of plasmid.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.1-14
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• 2004

More than twenty years have passed since the approval of the first recombinant DNA product for therapeutic use (recombinant human insulin, 1982). However, the biotechnology industry is still facing a shortage of manufacturing capacity due to the increasing demand of therapeutic proteins. This demand has prompted the search for a growing number of biological production systems but, nevertheless, the Gram-negative bacterium Escherichia coli remains one of the most attractive production hosts. This review highlights the most important features and developments of plasmid vector design, emphasizing the different reported strategies for improving the expression and secretion of heterologous proteins using the cellular machinery of E. coli.

• Applied Biological Chemistry
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• v.31 no.3
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• pp.267-273
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• 1988

Transformed, hybrid strains of the yeast Saccharomyces capable of simultaneous secretion of both glucoamylase and ${\alpha}-amylase$ have been produced. These strains can carry out direct, one-step assimilation of starch with conversion efficiency greater than 93% during a 5 day growth period. One of the transformants converts 92.8% of available starch into reducing sugars in only 2 days. Glucoamylase secretion by these strains results from expression of one or more chromosomal STA genes derived from Saccharomyces diastaticus. The strains were transformed by a plasmid(pMS12) containing mouse salivary ${\alpha}-amylase$ cDNA in an expression vector containing yeast alcohol dehydrogenase promoter and a segment of yeast $2{\mu}$ plasmid. The major starch hydrolysis product produced by crude amylases found in culture broths is glucose, indicating that ${\alpha}-amylase$ and glucoamylase act cooperatively.

• KSBB Journal
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• v.13 no.6
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• pp.649-655
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• 1998

Hantaviruses are rodent hosts-borne viruses belonging to the family Bunyaviridae, and are etiologic agents for two acute diseases, i.e., Haemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS). There have been a lot of reports on prophylactic vaccines and diagnostics for the diseases, but most of viral antigens have been prepared by eukaryotic cell culture. Nucleocapsid proteins of Hantaviruses are known as the major viral antigens. Thereby, we prepared nucleocapsid genes of Hantaan virus and Seoul virus by RT-PCR and cloned into plasmid vectors, pET-3a and pKK223-3. Both genes were expressed in Escherichia coli with higher expression level of Seoul viral nucleocapsid protein compared to that of Hantaan in pET-3a. Hantaan viral gene was expressed much higher level in plasmid pET-3a that in pKK223-3. About 30% of expressed nucleocapsid protein was soluble and the rest was remained in insoluble fraction.