• Environmental Analysis Health and Toxicology
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• v.29
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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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• v.13 no.6
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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.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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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.

• Animal cells and systems
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• v.5 no.3
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• pp.195-198
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• 2001

Hepatitis B virus (HBV) polymerase, which possesses the activities of terminal binding, DNA polymerase, reverse transcriptase and RNaseH, has been shown to accomplish viral DNA replication through a pregenomic intermediate. Because the HBV polymerase has not been purified, the expression of HBV polymerase was examined in an E. coli expression system that is under the regulation of arabinose operon. The expressed individual domain containing terminal binding protein, polymerase, or RNaseH turned out to be insoluble. The activities of those domains were not able to be recovered by denaturation and renaturation using urea or guanidine-HCI. The expressed reverse transcriptase containing the polymerase and RNaseH domains became extensively degraded, whereas the proteolysis was reduced in a Ion- mutant. These results indicate that Lon protease proteolyzes the HBV reverse transcriptase expressed in E. coli.

• Korean Journal of Microbiology
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• v.37 no.4
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• pp.245-252
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• 2001

Erm proteins, MLS (macrolide-lincosamide-streptogramin B) resistance factor proteins, show high degree of amino acid sequence homology and comprise of a group of structurally homologous N-methyltransferases. On the basis of the recently determined structures of ErmC` and ErmAM, ErmSF was divided into two domains, N-terminal end catalytic domain and C-terminal end substrate binding domain and attempted to overexpress catalytic domain in E. coli using various pET expression systems. Three DNA fragments were used to express the catalytic domain: DNA fragment 1 encoding Met 1 through Glu 186, DNA fragment 2 encoding Arg 60 to Glu 186 and DNA fragment 3 encoding Arg 60 through Arg 240. Among the pET expression vectors used, pET 19b successfully expressed the DNA fragment 3 and pET23b succeeded in expression of DNA fragment 1 and 2. But the overexpressed catalytic domains existed as inclusion body, a insoluble aggregate. To assist the soluble expression of ErmSF catalytic domains, Coexpression of chaperone GroESL or Thioredoxin and lowering the incubation temperature to $22^{\circ}C$ were attempted, as did in the soluble expression of the whole ErmSF protein. Both strategies did not seem to be helpful. Solubilization with guanidine-HCl and renaturation with gradual removal of denaturant and partial digestion of overexpressed whole ErmSF protein (expressed to the level of 126 mg/ι culture as a soluble protein) with proteinase K, nonspecific proteinase are under way.