• Journal of Life Science
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• v.15 no.6 s.73
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• pp.928-934
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• 2005

ATF2 is a cellular transcription factor which belongs to the CREB/ATF class and it is leucine zipper protein which generally binds to DNA as dimers. This paper presents the procedure for subcloning the ATF2 gene and the results of experiment used the expressed ATF2. The pET expression vector was used since it produced 6xHis fusion protein for easy purification using affinity column. The Nickel chelating chromatography was used for Purifying the expressed ATE2 from E- codi BL2l. Subsequen시y In vitro binding pull-down assay showed the binding specificity of ATF2 with AP-1 family factors such as BATF, c-Fos, c-Jun and ATF2 itselgf. ATF2 forms homodimer as well as strong heterodimer with BATF. It also forms stable dimer with c-Jun but barely binds with c-Fos.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.193-198
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• 2012

This study was to examine expression of the recombinant full-length adiponectin (recombinant adiponectin) in insect ovarian cell culture system and to characterize structural properties of the recombinant adiponectin secreted in medium. Gene construct encoding the recombinant adiponectin contained N-terminal collagen-like domain (110 Amino Acids, AAs), C-terminal globular domain (137 AAs) and C-terminal peptides for detection with V5 antibody (26 AAs included adaptor peptide) and purification using the 6xHis tag (6 AAs). The approximate molecular weight of the product (monomer) was 35 kDa. Molecular mass species of the expressed recombinant adiponectin were monomer (~35 kDa), dimer (~70 kDa), trimer (~105 kDa) and hexamer (~210 kDa). The major secreted species were the LMW forms, such as monomer, dimer, and trimer. There was MMW of hexamer as minor form. HMW multimers (~300 kDa) were shown as a tracer or not detected on the SDS-PAGE in several experiments (data not shown). The multimer forms in this study were not compatible to those in animal or human serum and adipose tissue by other researcher's study in which the major multimer forms were HMW. By protein denaturing experiments with reducing reagent (${\beta}$-MeOH), anionic detergent (SDS) and heat ($95^{\circ}C$) on the SDS-PAGE, not all adiponectin multimers seemed to have disulfide bond linked structure to form multimers. The recombinant adiponectin which expressed in insect ovarian cell culture system seemed to have the limitation as full physiological regulator for the application to animal and human study.

• The Plant Pathology Journal
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• v.26 no.1
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• pp.25-31
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• 2010

Papaya ringspot virus (PRSV) causes the most widespread and devastating disease in papaya. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The entire coat protein (CP) gene of Papaya ringspot virus-P biotype (PRSV-P) was amplified by RTPCR. The amplicon was inserted into pGEM-T vector, sequenced and sub cloned into a bacterial expression vector pRSET-A using a directional cloning strategy. The PRSV coat protein was over-expressed as a fusion protein in Escherichia coli. SDS-PAGE gel revealed that CP expressed as a ~40 kDa protein. The recombinant coat protein (rCP) fused with 6x His-tag was purified from E.coli using Ni-NTA resin. The antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified PRSV. The purified rCP was used as an antigen to produce high titer PRSV specific polyclonal antiserum. The resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay and compared its sensitivity levels with ELISA based assays for detection of PRSV isolates. IC-RT-PCR was shown to be the most sensitive test followed by dot-blot immunobinding assay (DBIA) and plate trapped ELISA.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.205-210
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• 2013

Lumazine protein is a fluorescent protein isolated from the bioluminescent bacteria of Photobacterium species. To generate minimal size of lumazine protein with possessing fluorescent characteristic, the gene coding for the wild type N-terminal domain of lumazine protein (N-LumP 118) containing amino acids up to 118 from Photobacterium leiognathi was produced. In addition, the genes coding for the variant proteins of N-LumP 118, replaced with one tryptophan amino acid (N-LumP 118 V41W, S48W, T50W, D64W, and A66W), were also constructed by Polymerase Chain Reaction and Site Directed Mutagenesis. These proteins were expressed in Escherichia coli by transformation with recombinant plasmids and purified by 6X-His tagging system. Spectroscopic studies have show that the purified proteins are capable of binding to the fluorescent ligand 6,7-dimethyl-8-ribityllumazine, resulted in showing of fluorescent characteristic with the minimal size of protein. From these studies, the mutant proteins containing single tryptophan amino acid residue, possessing its own intrinsic flouophore character at the different position, will be able to the use as a probe for further studies to deduce their three dimensional structure and the binding modes.

• Journal of Applied Biological Chemistry
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• v.54 no.2
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• pp.94-100
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• 2011

A gene encoding a putative phospholipase D was isolated from Bacillus licheniformis and cloned into pGEM-T easy vector. The gene was expressed in E. coli BL21 (DE3) using a pET-21(a) vector containing His6 tag. Affinity purification of the recombinant phospholipase D with nickel-nitrilotriacetic acid (Ni-NTA) resin resulted major one-band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The purified enzyme showed a molecular weight of 44 kDa. The optimum activity of enzyme was around pH 7.0 and the enzyme was also the most stable around this condition. The optimum temperature was about $40-45^{\circ}C$ and the enzyme still showed considerable activities at wide range of temperature. Among various detergents, Triton X-100 significantly increased the enzyme activity, resulting in 181% activity of control at 0.6 mM of the detergent. Calcium ion did not significantly affect the enzyme activity, suggesting that the enzyme might be classified into $Ca^{2+}$-independent PLD.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.317-323
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• 2016

An isoflavone glucosidase that catalyzes the hydrolysis of isoflavone glucosides into glucose and corresponding aglycones was purified from Candida fermentati SI. The N-terminal sequence was determined to be GLNCDYCN. We designed degenerate primers on the basis of these amino acid sequences and successfully cloned the full structural gene sequence of the isoflavone glucosidase using inverse PCR. The exo-β-(1,3)-glucanase gene consists of 1227 base-pair nucleotides, encoding a 408-amino-acid sequence that shares 41–96% amino acid homology with other yeast exo-β-(1,3)-glucanases belonging to glycoside hydrolase family 5. The recombinant exo-β-(1,3)-glucanase was expressed in Pichia pastoris X-33, using a pPICZA vector system, and further characterized. The molecular mass of the purified exo-β-(1,3)-glucanase was estimated by SDS-PAGE to be 47 kDa. The optimal pH and temperature were pH 4.5 and 40℃, respectively. The K_m values of the purified exo-β-(1,3)-glucanase for daidzin and genistin were 0.12 mM and 0.14 mM, respectively. The V_max values of the purified isoflavone glucosidase were 945.03 U/mg for daidzin and 835.92 U/mg and for genistin.