• Environmental Analysis Health and Toxicology
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• 제29권
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• pp.7.1-7.8
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• 2014

Objectives The present study was designed to systematically characterize the denaturation and the renaturation of double stranded DNA (dsDNA), which is suitable for DNA hybridization. Methods A series of physical and chemical denaturation methods were implemented on well-defined 86-bp dsDNA fragment. The degree of each denaturation was measured and the most suitable denaturation method was determined. DNA renaturation tendency was also investigated for the suggested denaturation method. Results Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. However probe sonication fully denatured DNA in 5 minutes. 1 mol/L sodium hydroxide (alkaline treatment) and 60% dimethyl sulfoxide (DMSO) treatment fully denatured DNA in 2-5 minutes. Conclusions Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments. Among chemical methods, 60% DMSO was the most adequate denaturation method since it does not cause full renaturation during DNA hybridization.

• Journal of Microbiology and Biotechnology
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• 제11권5호
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• pp.887-889
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• 2001

The gene encoding yeast pro-carboxypeptidase Y (pro-CPY) has been cloned and expressed in Escherichia coli. Most of the expressed pro-CPY was accumulated as cytoplasmic insoluble aggregates. In our previous study, active CPY was obtained by renaturation of entirely denatured pro-CPY followed by in vitro proteolytic processing with proteinase K along with the activation process. The same refolding process was performed to produce an active CPY from pro-CPY inclusion bodies with renaturation buffers containing proteinase K at different concentrations. The refolding efficiency decreased from $25\%\;to\;2\%$ in the renaturation buffers containing proteinase K at concentrations of $60{\mu}g/ml\;and\;0.6{\mu}g/mi$, respectively. In an attempt to increase the refolding efficiency with a lesser amount of proteinase K, a novel fed-batch refolding process was developed. In a fed-batch refolding, 99 ml of the renaturation buffer containing pro-CPY was gradually added into 1 ml of the renaturation buffer containing $60{\mu}g/ml$ of proteinase K to give a final proteinase K concentration of $0.6{\mu}g/ml$. The fed-batch refolding process resulted in a refolding efficiency of $18\%$, which corresponded to a 9-fold increase over that ($2\%$) in the batch process.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.989-992
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• 2003

Recombinant human tissue plasminogen activator deletion mutein (Reteplase) is a clinically promising thrombolytic drug. Reteplase cDNA was subcloned into a bacteria expression system, and the resultant recombinant was biologically characterized. The Reteplase was expressed in Escherichia coli as an inclusion body, and the downstream processes of the Reteplase inclusion body included denaturation, renaturation, and purification. A protein disulfide isomerase (PDI) was used to assist the refolding of Reteplase, and it was found to increase the refolding rate from less than 2％ to more than 20％. The refolded Reteplase was purified through two chromatography steps, including lysine-coupled agarose affinity chromatography and then CM-sepharose cation-exchange chomatography. The purity of r-PA was analyzed by Western bolt analysis, and N-terminal amino acid and amino acid composition analyses confirmed the end-product. Reteplase showed higher thrombolytic potency in an animal thrombus model.

• 미생물학회지
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• 제36권1호
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• pp.14-19
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• 2000

Penicillium oxalicum(HCLF-34)의 세포외 분비효소로부터 ultrafilration, gel filtration chromatograph와 anion exchange chromatography법을 이용하여 남조세균(Anabaena cylindrica) 분해효소를 분리하였다. 이 효소의 분자량은 약 22 kDa이며, renaturation SDS-PAGE에서 monomer로서 남조세균 분해 활성을 갖는다. 아미노산 서열은 N-말단부터$NH_(2)$-Glu-Ser-Tyr-Ser-Ser-Asn-Ala-Ala-Gly-Ala-Val-Leu-Ile---, 13개의 아미노산을 분석하였으며, 분석된 아미노산의 homology를 조사한 결과 aspergillopepsin II precursor(acid protease A)와 13개의 아미노산 중 11개(84%)의 유사도를 나타내었고, acid proteinase EapC precursor과 13개 중 10개(81%)의 유사도를 나타내었다.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2002년도 제37회 국제학술심포지움 및 추계학술대회
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• pp.9-17
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• 2002

Renaturation of Lysozyme by size exclusion chromatography(SEC) to improve yield as well as the initial and final protein concentration has been studied in detail, Although urea decreases the rate of proteins refolding, it can suppress protein aggregation to sustain pathway of correct refolding at high protein concentration, and there existed an optimum urea concentration in renaturation buffer. Lysozyme was successfully refolded from initial protein concentration of up to 100mg/m1 by SEC, the yield was more than 40%. And the refolding of Interferon-${\gamma}$ was further investigated.

• KSBB Journal
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• 제13권2호
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• pp.133-140
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• 1998

Using rlFN-$\alpha$ and rhGH as the model proteins, the refolding performances of the published processes were evaluated and compared. Key engineering parameters such as the type of denaturant and this concentration, protein concentration in the refolding buffer, and pH and ionic strength of the buffer were experimentally investigated. Furthermore, the role of a co-solvent of surfactant type in aggregation reduction was also studied. Of the denaturants tested (8M urea, 6M guanidine HCI, 0.5% SDS), SDS at alkaline pH (9.5) and ambient temperature gave the highest recovery yield. The SDS process was effective in the refolding of observed where dissolution proceeded better under lower strength (10 mM) but aggregation was suppressed under higher strength (>50 mM.) When PEG-4000 and/or Tween were added as co-solvent or refolding-enhancing additive, 1.6-2 times higher yield was realized. The‘masking’of the hyrophobic patches located on the surface of the protein with the surfactant molecules was believed to be responsible for the considerable reduction in aggregation during refolding.

• BMB Reports
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• 제39권2호
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• pp.150-157
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• 2006

Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also non-native intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cystiene forms are active in radioreceptor binding assay.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1996년도 정기총회 및 학술발표회
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• pp.27-27
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• 1996

SecA protein which has a pivotal role in the preprotein cranslocation across the inner membrane of Escherichia coli is a water-soluble protein with an unusual property of penetrating the membrane readily. An interesting and important question is what structural characteristics of SecA enables its ready penetration of lipid bilayer. The conformational properties of SecA in solution at 3$0^{\circ}C$, pH 7.5 were observed by hydrogen-tritium (HT) exchange, and denaturant-induced denaturation/renaturation and thermal unfolding. (omitted)

• 미생물학회지
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• 제35권3호
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• pp.231-236
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• 1999

Penicillium oxalicum 으로부터 세포외로 분비되는 Anabaena cylindrica 분해효소의 분자량은 renaturation SDS-PAGE에서 약 22kDa 으로 확인되었으며, 분해 효소의 농축은 ultrafiltration cut off fraction 중 30-10 kDa 구간에서 수획하였다. 최적 활성조건의 측정 결과 적정 pH는 3.5-4.0, 적정반응 온도는 $20^{\circ}C$, 그리고 온도 안정성은 $4^{\circ}C$ 이하에서 100% 이상, 20-$90^{\circ}C$ 범위에서는 50% 이상의 활성을 나타내었다. 금속이온 및 효소안정제의 영향에서는 $Na^+$,$K^+$, $Ba^(2+)$, $Mg^(2+)$, $Mn^(2+)$의 양이온과 BSA는 효소의 활성을 촉진시키는 반면, $Ca^(2+)$, $Cu^(2+)$의 양이온과 EDTA, PMSF 는 효소의 활성을 억제하는 작용을 하였다. 이러한 금속이온과 안정제의 영향에서 1가, 2가 양이온에 의해 활성이 증가하고, $Fe^(3+)$, $Ca^(2+)$, $Cu^(2+)$의 양이온에 의해서는 활성이 감소하는 결과는 대부분의 세포벽 분해효소가 갖는 특성과 유사한 결과였다. 분해효소는 A. cylindrica 과 Micrococcus. luteus 의 세포벽을 기질로 사용한 효소의 활성 반응에서 반응 시작 후 1시간에서 5시간 사이에 반응 산물로 환원당의 양이 급격히 증가하였다.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.474-481
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• 1996

Since the commercially available rabbit anti-cyclin D3, generated from c-terminal 16 amino acid residues which are common to human and murine cyclin D3, is highly cross-reactive with many other cellular proteins of mouse, a new rabbit polyclonal anti-cyclin D3 has been raised by using murine cyclin D3 protein expressed at a high level in Escherichia coli as the immunogen. To express murine cyclin D3 protein in E. coli, the cyclin D3 cDNA fragment encoding c-terminal 236 amino acid residues obtained by polymerase chain reaction (PCR) was inserted into the NcoI/BamHI site of protein expression vector, pET 3d. Molecular mass of the cyclin D3 overexpressed in the presence of IPTG (Isopropyl $\beta$-D-thiogalactopyranoside) was approximately 26 kDa as calculated from the reading frame on the DNA sequence, and the protein was insoluble and mainly localized in the inclusion bodies that could be easily purified from the other cellular soluble proteins. When renaturation was performed following denaturation of the insoluble cyclin D3 protein in the inclusion bodies using guanidine hydrochloride, 4.4 mg of soluble form of cyclin D3 protein was produced from the transformant cultured in 100ml of LB media under the optimum conditions. Four-hundred micrograms of the soluble form of cyclin D3 protein was used for each immunization of a rabbit. When the antiserum obtained 2 weeks after tertiary immunization was applied to Western blot analysis, it was able to detect 33 kDa cyclin D3 protein in both murine lymphoma cell line BW5147.G.1.4 and human Jurkat T cells at 3,000-fold dilution with higher specificity to murine cyclin D3, demonstrating that the new rabbit polyclonal anti-murine cyclin D3 generated against c-terminal 236 amino acid residues more specifically recognizes murine cyclin D3 protein than does the commercially available rabbit polyclonal antibody raised against c-terminal 16 amino acids residues.