• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.115-118
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• 2000

Recombinant plasmids harboring a heterologous gene coding for the enhanced green fluorescent protein (EGFP) were transfected and expressed in Drosophila melanogaster S2 cells. A stable transformation of polyclonal cell populations expressing EGFP were isolated after 4 weeks of selection with hygromycin B. The recombinant EFGP expressed in transformed S2 cells consisted of a molecular weight of 27 kDa. EGFP expression was also confirmed by fluorometric measurement. The maximum EGFP concentration was about 9.3 mg/I. The present findings demonstrate not only the successful stable expression of EGFP in Drosophuila was about 9.3 mgI. The present findings demonstrate not only the successful stable expression of EGFP in Drosophila S2 cells, but also the use of EGFP as a reporter to analyze gene expression, with its potential of a Drosophila cell expression system for recombinant protein production being an alternative to a baculovirus-insect cell expression system.

• BMB Reports
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• 제39권1호
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• pp.55-60
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• 2006

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of ${\alpha}_1$-antitrypsin (${\alpha}_1AT$) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at $37^{\circ}C$). Once the ${\alpha}_1AT$ moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint ($C_m$) of the ${\alpha}_1AT$ variant, around 20% of the phage retained binding affinity to anti-${\alpha}_1AT$ antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the ${\alpha}_1AT$ variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.

• BMB Reports
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• 제30권1호
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• pp.21-25
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• 1997

The growth. freezing resistance and electrophoretic protein patterns of carrot callus cultures were investigated following treatment with NaCl for various' intervals at 20$^{\circ}C$. Following 7 day exposure to 250 mM NaCl. freezing tolerance increased, which was measured by 2.3.5-triphenyl tetrazolium chloride (TTC) assay and fresh weight was reduced compared to control cells. Changes of electrophoretic patterns of total and boiling stable proteins were investigated using one or two dimensional gel system. Several proteins with molecular weight of 43 and 21 kDa increased by NaCl treatment. The most prominent change was detected in 21 kDa protein. The steady state level of this protein increased in NaCl treated cells, but decreased in control cells. Twenty one kDa protein was detected only in the NaCl treated cell when boiling stable protein was analyzed. The isoelectric point of 21 kDa protein was identified as 5.7. The timing of increase of 21 kDa protein was correlated to freezing resistance which implied the role of this protein in the induction of freezing resistance of the cell.

• Journal of Microbiology and Biotechnology
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• 제23권6호
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• pp.872-877
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• 2013

In a previous study, we isolated and reported a second species of the Saccharophagus genus, Saccharophagus sp. strain Myt-1. In the present study, an alginate lyase gene (algMytC) from the genomic DNA of Myt-1 was cloned and characterized. The DNA sequence fragment obtained contained an open reading frame of 1,032 bp that encoded a protein of 343 amino acids with an estimated molecular mass of 37.6 kDa and a pI of 6.60. The deduced protein, AlgMytC, had the conserved amino acid sequences (RTELREM, QIH, YFKAGVYNQ) of the polysaccharide lyase family 7. A BLAST homology search indicated that AlgMytC shared an amino acid sequence identity of 95.9% with alg7A of S. degradans 2-40. The cloned and purified AlgMytC protein showed optimal activity at $40^{\circ}C$, and retained more than 90% of its total activity even after treatment at $25^{\circ}C$ for 24 h. AlgMytC was very alkaliphilic with an optimal pH of 9.0, and more than 90% of its activity was retained in the pH range 8.5-10.0. Moreover, AlgMytC was stable over a wide pH range. The activity of AlgMytC was also stable in the presence of various detergents.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.805-810
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• 2000

An antifungal protein antagonistic to the rice blast fungus, Pyricularia oryzae was purified from Paenibacillus macerans PM-1 by ammonium sulfate fractionation, Q Sepharose Fast Flow column chromatography, Phenyl Sepharose CL-4B column chromatography and Superose 12 gen filtration. An apparent molecular mass of the purified antifungal protein was determined as 8 kDa by SDS-PAGE and 9 kDa by analytical gel filtration, respectively, suggesting that the purified protein is a monomer. The antifungal protein was stable at pH range from 7-12 and up to $100^{\circ}C$. The protein was also stable at 0.1-1% Tween 20 and Triton X-100. The N-terminal amino acid sequence of the antifungal protein was Thr-Glu-Leu-Pro-Leu-Gly-Ile-Val-Met-Asp-Lys-Tyr-Thr-Asp-Ala-Phe-Lys-Phe-Asp-Met-Phe. Comparison of the determined sequence with other peptide and DNA sequences did not reveal homology at all. Therefore, the purified antifungal protein was speculated to be a novel protein. The condidial germination in vitro of P. oryzae KJ301:93-39 by the purified protein ($5.9{\mu} g/ml$) was limited to $9{\pm}3.2%$ only, compared with $69{\pm}2.4%$ of the control. Ungerminated conidia were swollen at basa and mid cell by the purified protein. In vivo bioassay for inhibition of conidial germination of P. oryzae KJ 301, one of the most predominating racesin Korea. the purified protein ($5.9{\mu} g/ml$)strongly inhibited the conidial germination. The conidia, even though germinated, could not develop any further to produce appressoria efficiently.

• 한국수산과학회지
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• 제28권6호
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• pp.728-735
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• 1995

The peptidyl prolyl cis-trans isomerase(PPlase, EC 5.2.2.8) from Bacillus stearothermophilus SIC1 was extracted from the cells treated with by lysozyme. PPlase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration (FPLC). The purity of purified the enzyme after Superose 12 column chromatography was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPlase was estimated as 18,000 by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0 to 8.0. The enzyme was considerably stable after heat treatment at $60^{\circ}C$ for 30 minutes, and the enzyme was quite stable up to $65^{\circ}C$. The presence of the PPlase in the refolding solution accelerated the isomerization rate of the assay peptide.

• BMB Reports
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• 제41권10호
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• pp.716-721
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• 2008

To explore the effects of deregulated expression of the EBNA1 binding protein 2 (EBP2) on cell growth, we generated human HEK293 stable clones constitutively expressing an EBP2-EGFP fusion protein. We found both RNA and protein levels of cyclin E1, a dominant oncoprotein, were elevated in the EBP2- EGFP stable clones. These findings were confirmed by flow cytometry bivariate analysis of cyclin expression versus DNA content. Moreover, the increase in p21 expression and the specific phosphorylation at Ser1981 of ATM and Ser15 of p53 were also observed in these stable clones, and these observations may explain the failure to observe an increase in Cdk2 kinase activity. In addition, after one year of passage culture, the EBP2-EGFP stable clones tended to lose 4 to 5 chromosomes per cell when compared to that of control cells. All of these findings provide a possible link between deregulated expression of EBP2 and tumor development.

• BMB Reports
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• 제35권6호
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• pp.629-636
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• 2002

Protein kinase CKII (CKII) is required for progression through the cell division cycle. We recently reported that the $\beta$ subunit of protein kinase CKII ($CKII{\beta}$) associates with CKBBP1 that contains the Ring-H2 finger motif in the yeast two-hybrid system. We demonstrate here that the Ring-H2 finger-disrupted mutant of CKBBP1 does not interact with purified $CKII{\beta}$ in vitro, which shows that the Ring-H2 finger motif is critical for direct interaction with $CKII{\beta}$. The CKII holoenzyme is efficiently co-precipitated with the wild-type CKBBP1, but not with the Ring-H2 finger-disrupted CKBBP1, from whole cell extracts when epitope-tagged CKBBP1 is transiently expressed in HeLa cells. Disruption of the Ring-H2 finger motif does not affect the cellular localization of CKBBP1 in HeLa cells. The increased expression of either the wild-type CKBBP1 or Ring-H2 finger-disrupted CKBBP1 does not modulate the protein or the activity levels of CKII in HeLa cells. However, the stable expression of Ring-H2 finger-disrupted CKBBP1 in HeLa cells suppresses cell proliferation and causes the accumulation of the G1/G0 peak of the cell cycle. The Ring-H2 finger motif is required for maximal CKBBP1 phosphorylation by CKII, suggesting that the stable binding of CKBBP1 to CKII is necessary for its efficient phosphorylation. Taken together, these results suggest that the complex formation of $CKII{\beta}$ with CKBBP1 and/or CKII-mediated CKBBP1 phosphorylation is important for the G1/S phase transition of the cell cycle.

• Journal of Microbiology and Biotechnology
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• 제7권2호
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• pp.132-137
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• 1997

A novel protein (HEAAE, High Essential Amino Acid Encoding Protein), rich in essential amino acids ($75{\%}$ of total), was designed and constructed in our laboratory. The designed peptides were analyzed by SYBLE and stable secondary and tertiary structures were predicted. The monomeric form (HEAAE-1) of the protein consists of 20 amino acid residues with four additional amino acids comprising a potential ${\beta}$-turn (HEAAE-4). Size exclusion analysis demonstrated that the monomer is self-aggregates in aqueous solution to form higher ordered multimeric structures, which are very reminiscent of natural plant storage proteins. The DNA encoding this amino acid sequence was synthesized, and from this monomeric gene fragment (heaae-1), the stable tetrameric form of the gene (heaae-4) was generated by subcloning into the E. coli expression vector pKK223-3. A clear 6 kDa polypeptide band corresponding to the molecular weight of the dimeric form (HEAAE-2) was detected. The smeared band which appeared around the molecular weight corresponding to HEAAE-4 of 11 kDa suggested that the tetramer form of this protein might be processed into smaller size products.

• 한국식품과학회지
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• 제32권4호
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• pp.822-827
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• 2000

새우 알러지를 감소시키기 위한 방법으로써 감마선 조사기술의 이용 가능성을 평가하였다. 새우로부터 분리한 열안정성 단백질(HSP)과 병원에서 임상진단용으로 사용하는 새우 단백질 추출액을 1, 3, 5, 7, 10 kGy의 선량으로 감마선을 조사하였다. 단백질 추출액 및 HSP의 알러지성과 항원성의 변화를 mAb와 환자의 IgE를 사용한 ELISA법과 immunoblotting법으로 측정하였고, 단백질의 구조변화는 gel permeation chromatography와 SDS-PAGE로 관찰하였다. 환자의 IgE는 감마선 조사된 단백질 추출액과 HSP용액 모두에서 감마선 조사선량이 증가함에 따라 감소하였다. GPC-HPLC와 sandwich ELISA법으로 HSP를 정량하였을 때도 역시 감마선 조사선량이 증가함에 따라 감소하였다. SDS-PAGE pattern 변화에서 HSP는 감마선 조사에 의해 변화되어 더 큰 분자량으로 전환되었다. 이러한 결과는 감마선 조사가 새우 알러지 억제에 적용 할 수 있다는 가능성을 시사하였다.