• The Journal of Natural Sciences
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• v.9 no.1
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• pp.19-24
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• 1997

Using the PCR(polymerase chain reaction method), gone 1 of phage K11 coding for K11 phage RNA polymerase has been cloned and expressed under the control of lac promoter. K11 phage RNA polymerase was conventionally purified through the DEAE-sephacel and Affigel blue column chromatographies. The 0.2-0.3 M $NH_4Cl$ fractions of DAEA-sephacel column chromatography showed K11 phage RNA polymerase activity and further purification with Affigel blue column chromatography showed nearly single protein band on SDS-polyacryl amide gel. K11 phage RNA polymerase, which is one of the T7 group phage RNA polymerase (E. coil phage T7, T3 and Salmonella tyhimurium phage SP6 RNA polymerase), shares high degrees of homology with the other T7 group phage RNA polymerase. Previously we constructed T7 and SP6 promoter variants and revealed promoter specificity of T7 and SP6 RNA polymerase (Lee and Kang, 1993). To investigate the promoter specificity of K11 RNA polymerase in vitro K11 promoter activity was measured with SP6 promoter variants. The SP6 promoter variant share highest degrees of sequence homology with K11 promoter sequence show strongest promoter activity.

• The Microorganisms and Industry
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• v.14 no.1
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• pp.7-11
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• 1988

Efficient in vitro RNA synthesis can be easily accomplished from cloned DNA using bactrio-phage SP6, T7 or T3 RNA polymerase. Despite its popularity as in vitro transcription system, molecular mechanisms of bacteriophage transcription has not been studied, although physical and catalytic properties of several phage RNA polymerases have well been documented (1). Only recently the T7 promoter has been physically mapped by footprinting of the T7 RNA polymerase (2,3). These simple phage systems, however, could be useful for detailed molecular studies of transcription.

• Food Science and Biotechnology
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• v.16 no.3
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• pp.398-403
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• 2007

The effectiveness of phage biocontrol depends on the activity of bacteriophage in a given environment. In order to investigate the infectivity and the stability of bacteriophages in representative environments, three virulent phages, Listeria phage A511, Salmonella phage Felix O1, and Escherichia coli phage T7, were subjected to different temperatures, pHs and salt concentrations (NaCl). Phage infectivity was also determined in the presence of divalent cations ($Mg^{2+}$ or $Ca^{2+}$). As a result, three phages exhibited a wide range of survival rates under various environments. Phage infectivity was directly correlated with bacterial growth under the applied conditions. One exception was Felix O1 that did not kill Salmonella grown in low pH (4.5). The failure was attributed to defective adsorption of Felix O1. This finding is significant as it provides an explanation for the inefficient phage biocontrol. Therefore, such information is crucial to improve phage biocontrol of pathogens.

• The Journal of Natural Sciences
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• v.14 no.1
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• pp.51-61
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• 2004

T7 RNA polymerase is a single subunit RNA polymerase able to accomplish whole transcription process without auxiliary factors. T7 phage lysozyme involcing in destruction of host cell wall repress T7 transcription and affects transcription termination process. Therefore expression vector pT7lys containing T7 phage lysozyme gene was constructed and expressed. T7 phage lysozyme protein was purified to homogeneity by Ni-NTA column chromatography. Also amidase activity of the purified lysozyme was identified. In order to understand the effect of the lysozyme on the sequence specific transcription termination. T7 transcription elongation complexes at the site rrnB T1 transcription termination signal were made in the presence the lysozyme. The results shows that the transcription elongation complexes are unstable in the presence of T7 phage lysozyme.

• Korean Journal of Microbiology
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• v.17 no.3
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• pp.97-130
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• 1979

Many industrial processes those employ bacteria are subjected to phage infestations. In L-glutamic acid fermentions using acetic acid, the phage infestations of the organisms have been recently recognized. In efforts to elucidate the sources of phage contamination involved in the abnormal fermentation, a series of study was conducted to isolate the phages both from the contents of abnormally fermented tanks and the soil or sewage samples from the surroundings of a fermentation factory, to define major charateristics of the phage isolates, and finally to determine the correlation between the phage isolates and temperate phages originating from the miscellaneous bacterial species isolated from the soil or sewage samples. The results are summarized as follows; 1) All phages were isolated from the irregular fermentation tanks and soil or sewage samples, and they were designated as phage PR-1, PR-2, PR-3, PR-4, PR-5, PR-6, and PR-7, in the order of isolation. These PR-series phages were proved to be highly specific for the variant strains of Br. lactofermentum only, namely, phage PR-1 and PR-2 for Br. lactofermentum No. 468-5 and phage PR-3~PR-7 for Br. lactofemrentum No. 2256. By cross-neutralization test, the 7 phagescould be subdivided into 3 groups, i. e., phage PR-I and PR-2 the first, phage PR-3, PR-4, PR-5, PR-6 the second, and the phage PR-7 the third. 2) The 7 phages were virulent under the experimental conditions. They produced plaques with clear and relatively sharp margins without distinct halo. The mean sizes of plaques were 1.5mm in diameter for phage PR-1 and PR-2, and 1. Omm for phages PR-3~PR-7. Double layer technique modified by Hongo and described by Adams, was applied to assay of the PR-series phages. The factors influencing the plaques were as follows;young age cells of host bacteria cultured for 3-6 hours represented the largest number and size, optimum was pH 7.0, incubation temperature was $30^{\circ}C$, and agar concentration and amount of overlayer medium were 0.6% and 0.2ml, respectively. 3) PR-series phages were stable in 0.05M tris buffer and 0.1M ammonium acetate buffer solution. The addition of $5{\times}10^{-3}M$ magnesium ion effectively increased the stability. Thermostability experiments indicated that PR-series phages were stable at the teinperture between $50^{\circ}{\sim}55^{\circ}C$ in nutrient medium, $45^{\circ}{\sim}50^{\circ}C$ in buffer solution. However, the phages mere completely inactivated at 603C and 65$^{\circ}$C within 10 minutes. The phages were stable at the range of pH6~9 in nutrient medium and of pH 8-9 in buffer solution, respectively. Exposure of the phages to UV for 25, 60 and 100 seconds resulted in the complete loss of infectivily, respectively. 4) Electron microscopy showed that PR-series phage particles exhibited rather similar morphology, differing in the size All of PR-series phages had a multilateral head and had a simple long tiil about three to five times long as compared with head. By the size, phage PR-1 and PR-2, PR-3, PR-4, PR-5, and PR-6 and PR-7 were classified into same groups, respectively. The head and tail size of phage PR-1, PR-5, PR-5(T) and PR-7 were 85nm, 74nm and 235nm and 350mm, and 72nm and 210nm, respectively. 5) Nucleic acids of PR-series phages were double stranded DNA. The G+C contents of phage PR-1, PR-5 and PR-7 were 56.1, 52.9 and 53.7, respectively. The values of G+C contents derived from the $T_m$ were in agreement with the chemically determined values. 6) PR-series phages effectively adsorbed on their host bacteria at the rate of more than 90% during 5 min. K value for phage PR-1, PR-5 and PR-7 were calculated to be $6{\times}10^9 ml$ per minute, respectiveky. The pH of the medium did effect adsorption rate, but both temperature and age of host cells did not. Generally, optimum adsorption condition of phages seemed to be almost same as optimum growth conditions of host bacteria. 7) In one-step growth experiments, the latent periods at $30^{\circ}C$ for PR-1, and PR-7 were about 70, 50 and 55 min, respectively. The corresponding average burst size was 200, 70 and 90, respectively. Lpsis period according to the multiplicity of infection and a phage series. In case of m. o. i. 100, strain No. 2256 (PR-5) and No. 468-5(PR-1) failed to grow and turbidity decreased after 50 and 70min, respectively. 8) In the lysate of a plaque purified phage PR-5 infected bacteria, there observed 2 types ofphage particles, i. e., phage PR-5 and PR-5 (T) of similar morphology but differing at the length of phage tail, and phage tail like particles. The phage taillike particles could be divided into 4 types by the length. Induction experiments of Br. lactofermentum with UV irradiation, mitomycin C or bacitracin treatment produced neither phage PR-5 (T) or phage tail-like particles. 9) No lysis occured when the growth of 7 strains of miscellaneous bacteria, isolated from soil and sewage samples, were inoculated with either phage PR-5 (T) or phage tail-like particles the inoculation of phage PR-5 pellet resulted in the growth inhibition of the orgainsms in the spot test. The lysates obtained from 3 miscellaneous soil derived bacteria following mitomycin C treatment the growth of Br. lactofermentum, but did not lyze the bacterium.

• The Journal of Natural Sciences
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• v.14 no.2
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• pp.83-91
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• 2004

Recently phage K11 lysozyme was cloned and characterized in our lab. The K11 lysozyme was identified to have dual functions. It not only cuts a peptidoglycan bond in bacterial cell wall but also acts as an inhibitor of K11 RNA polymerase. It has been known that the T7 lysozyme binds specifically to T7 RNA polymerase and inhibits transcription. The dual activities of K11 lysozyme are atreeable to the case of T7 phage lysozyme and RNA polymerare. In order to identify the binding magnitude of K11 lysozyme with K11 RNA polymerase, yeast two-hybrid system was used. K11 phage lysozyme gene was introduced into pLexA plasmid and used as a prey. Also, K11 phage RNA polymerase gene was introduced into pJG4-5 and used as a bait. The binding between K11 lysozyme and K11 RNA polymerase was demonstrated by expression of reporter genes such as lacZ and leu2.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.2
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• pp.209-218
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• 1995

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, has been implicated in T7 DNA replication, recombination, and repair. Purified gene 2.5 protein has been shown to interact with the phage encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phage that contain null mutants of gene 2.5 were constructed by homologous recombination. These mutant phage $(T7\Delta2.5)$ cannot grow in Escherichia coli. After infection of E. coli with $T7\Delta2.5$, host DNA synthesis is shut off, and $T7\Delta2.5$ DNA synthesis is reduced to less than $1\%$ of wild-type phage DNA synthesis (Kim and Richardson, 1993, Proc. Natl. Aca. Sci. USA, 90, 10173-10177). A truncated gene 2.5 protein $(GP2.5-\Delta21C)$ deleted the 21 carboxyl terminal amino acids was constructed by in vitro mutagenesis. $GP2.5-\Delta21C$ cannot substitute for wild-type gene 2.5 protein in vivo; the phage are not viable and exhibit less than $1\%$ of the DNA synthesis observed in wild-type phage-infected cells. $GP2.5-\Delta21C$ has been purified to apparent homogeneity from cells overexpressing its cloned gene. Purified $GP2.5-\Delta21C$ does not physically into「act with T1 gene 4 protein as measured by affinity chromatography and immunoblot analysis. The mutant protein cannot stimulate T7 gene 4 protein activity on RNA-primed DNA synthesis and primer synthesis. These results suggest that C-terminal domain of gene 2.5 protein is essential for protein-protein interactions.

• Journal of Life Science
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• v.13 no.4
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• pp.541-550
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• 2003

E2 glycoprotein of hepatitis C virus (HCV) comprises a surface of viral particle together with E1 glycoprotein, and is thought to be involved in the attachment of HCV viral particle to receptor (s) on the permissible cells including hepatocytes, B cells, T cells, and monocytes. We constructed a phage library expressing cellular proteins of hepatocytes on the phage surface, which turned out to be 8.8${\times}$$10^5$ cfu of diversity and carried inserts in 95% of library. We screened both cDNA phage library and 12-mer peptide library to identify the cellular proteins binding to E2 protein. Some intracellular proteins including tensin and membrane band 4.1 which are involved in signal transduction of survival and cytoskeleton organization, were selected from cDNA phage library through several rounds of panning and screening. On the contrary, membrane proteins such as CCR7, CKR-L2, and insulin-like growth factor-1 receptor were identified through screening of peptide library. Phages expressing peptides corresponding to those membrane proteins were bound to E2 protein specifically as determined by neutralization of binding assay. Since it is well known that HCV can infect T cells as well as hepatocytes, we examined to see if E2 protein can bind to CCR7, a member of C-protein coupled receptor family expressed on T cells, using CCR7 transfected tells. Human CCR7 cDNA was cloned into pcDNA3.1(-) vector and transfected into human embryonic kidney cell, 293T, and expressed on the surface of the cell as shown by flow cytometer. Binding assay of E2 protein using CCR7 transfected cells indicated that E2 protein bound to CCR7 by dose-dependent mode, giving rise to the possibility that CCR7 might be a putative cellular receptor for HCV.

• BMB Reports
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• v.33 no.3
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• pp.242-248
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• 2000

Phage display of proteins is a powerful tool for protein engineering since a vast library of sequences can be rapidly screened for a specific property. In this study, we develop da new phage display vector that was derived from a pET-25b(+) vector. The pET-25b(+) was modified in order that the expressed protein would have a T7-tag at the amino terminus and GpS (a major coat protein of M13 phage) at the carboxyl terminus. Another vector without the gp8 gene was also constructed. The newly developed phagemid vectors have several advantageous features. First, it is easy to examine whether or not the target proteins are functional and faithfully transported into the periplasmic space. This feature is due to the fact that recombinant proteins are produced abundantly in the pET system. Second, the T7-tag makes it possible to detect any target proteins that are displayed on the surface of filamentous bacteriophage. To verify the utility of the vector, the clones containing the glutathione S-transferase (GST) gene as a target were examined. The result showed that the GST produced from the recombinant vector was successfully transported into the periplasmic space and had the anticipated enzyme activity. Western blot analysis using a T7-tag antibody also showed the presence of the target protein displayed on the surface of the phage. The phages prepared from the recombinant clones were able to bind to glutathione-Sepharose and then eluted with glutathione. These results showed that the new vectors developed in this study are useful for the phage display of proteins.

• Microbiology and Biotechnology Letters
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• v.18 no.3
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• pp.221-226
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• 1990

To detect prophages and bacterioeins, twenty strains of Bacillus circulans were treated with mitomycin C. The resulted lysates were subjected to electron microscopy, and also examined for killing and plaque-forming activities. Fifteen strains showed killing activity on two or more strains of Bacillue circulans. Killing agents were centrifuged in linear 5 to 20% sucrose gradient, and studied with electron microscopy which revealed the presence of particles.They looked morphologically like phage tail of 190 nm long with fiber (FA9, FA5) or without fiber (FA1, FA6), T even phage-like particle with a head of 50 nm in diameter and a tail of 140 nm long (FA7), or T7 phage-like particle with a head of 70 nm in diameter and a tail of 20 nm long (FA17). The killing agent of FA17 showed phage-forming activity on several strains different from killing sensitive strains of Bacillus circulans.