• Journal of Aquaculture
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• v.10 no.4
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• pp.425-433
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• 1997

The isolated soluble eye proteins from six species (Sepia esculenta, Sepiella japonica, Loligo chinensis, Todarodes pacificus, Loligo beka and Ocotopus minor) of class Cephaloopoda were compared by crossed immunoelectrophoresis methods using antibodies directed against total soluble eye proteins antigens. It showed a distinct antigen-antibody reaction between the species of order Sepioidea and various antiserum of class Cephalopoda. Our results suggested that the common precipitation lines of Sepia and Loligo species which were different from that of Octopus minor. By which obtained from elution of gel filtration chromatography and 12.5% SDS-polyacrylamide gel electrophoresis, the molecular weight of Todarodes pacificus eye protein (crystallin) was determined in abut 20-35 KDa.

• Journal of Industrial Technology
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• v.28 no.B
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• Korean Chemical Engineering Research
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• v.47 no.5
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• pp.624-629
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• 2009

It is the most important step to screen soluble and insoluble proteins when we attempt to improve the solubility of recombinant proteins through directed evolution approach. Here we show that the solubility of a recombinant protein in vivo can be examined by expressing the recombinant protein with beta-lactamase as a fusion partner. First we constructed an expression system which can produc a fusion protein with the C-terminal of beta-lactamase. Two soluble proteins, i.e. adenine deaminase and aspartate aminotransferase, and insoluble GlcNAc-2-epimerase were cloned into the developed expression vector, respectively. We investigated the effect of the expression of the three recombinant fusion proteins on the growth of E. coli, and confirmed that the solubilities of the recombinant proteins correlated with cell growth rates.

• Journal of Plant Biology
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• v.37 no.2
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• pp.151-158
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• 1994

Aleurone layers of normal and vp1 mutant maize kernels were extracted and centrifuged at 100,000g to yield a cytosol fraction. Binding of [3H]ABA cis, trans (＋)ABA to a soluble macromolecular components present in the cytosol was demonstrated by Sephadex chromatography and non-denaturing PAGE. The binding component was of high molecular weight and seems to be an aggregate of proteins. A rapid DEAE-cellulose filter method for assaying bound [3H]ABA to a soluble protein was adapted. Binding assays were performed with cytosol that had been preheated or incubated with several enzymes, indicating that heat and protease treatments disrupted the binding. This suggested that binding occurred to proteins. Some properties of the ABA binding proteins were described. The [3H]ABA binding were reduced dramatically when unlabeled ABA was added as a competitor, suggesting a specific binding of [3H]ABA. Gel filtration profiles and autoradiogram of [3H]ABA binding showed no difference in the binding components of Vp1 and vp1/vp1 mutant cytosol, indicating that Vp1 protein is not a sole ABA binding protein.

• Journal of Life Science
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• v.9 no.2
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• pp.146-152
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• 1999

In order to elucidate a role of residue 24 in the folding of tryptophan synthase $\alpha$ subunit, mutant proteins in which Thr 24 was replaced by Met, Ala, Ser, Leu or Lys were overexpressed in E. coli, and the extents of accumulated proteins as soluble or aggregated forms were examined. The mutant proteins with Met or Leu at residue 24 were predominantly accumulated as soluble forms as the native protein. On the other hand, mutant proteins with Ser, Ala or Lys at residue 24 were expressed as aggregated forms as well. This result suggests that residue 24 of tryptophan synthase $\alpha$ subunit may be implicated in the folding of this protein.

• Korean Journal of Food Science and Technology
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• v.39 no.4
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• pp.438-446
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• 2007

The fermented soybean product, cheonggukjang, is favored by many people, partly due to its bio-functional ingredients. Since the fermentation process of cheonggukjang is mediated by enzymes, including proteases, produced by microbes, analysis of the proteome profile changes in cheonggukjang during fermentation would provide us with valuable information for fermentation optimization, as well as a better understanding of the formation mechanisms of the bio-functional substances. The soluble proteins from cheonggukjang were prepared by a phenol/chloroform extraction method, in order to remove interfering molecules for high resolution 2-D gel analysis. Proteomic analysis of the cheonggukjang different fermentation periods suggested that most of the soluble soy proteins were degraded into smaller forms within 20hr, and many microbial proteins, such as mucilage proteins, dominated the soluble protein fraction. The proteomic profile of cheonggukjang was very different from natto, in terms of the 2-D gel protein profile. Among the separated protein spots on the 2-D gels, 50 proteins from each gel were analyzed by MALDI-TOF MS and PMF for protein identification. Due to database limitations with regard to soy proteins and microbial proteins, identification of the changed proteins during fermentation was restricted to 9 proteins for cheonggukjang and 15 for natto. From de novo sequencing of the proteins by a tandem MS/MS, as well as by database searches using BLASTP, a limited number of proteins were identified with low reliability. However, the 2-D gel analysis of proteins, including protein preparation methods, remains a valuable tool to analyze complex mixtures of proteins entirely. Also, for intensive mass spectrometric analysis, it is also advisable to focus on a few of the interestingly changed proteins in cheonggukjang.

• Proceedings of the Zoological Society Korea Conference
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• 1998.04a
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• pp.51.1-51
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• 1998

No Abstract, See Full Text

• Journal of Pharmaceutical Investigation
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• v.30 no.2
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• pp.73-83
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• 2000

Polyethylene glycol (PEG) is a water soluble, biocompatible, non-toxic polymer and PEGylation is a well established technique for the modification of therapeutic proteins and peptides. PEG-protein drugs have been extensively studies in relation to therapies for various diseases: cancer, inflammation and others. The covalent attachment of PEG to proteins and peptides prolonged plasma half-life, reduced antigenicity and immunogenicity, increased thermal and mechanical stability, and prevented degradation by enzymes. Several chemical groups for general and site specific conjugation have been exploited to activate PEG for amino group, carboxyl group, and cysteine groups. PEGylation of many proteins and peptides have been studied to enhance their properties for the potential uses. Also, the different positional isomers in several PEG-proteins have shown the difference in vivo stability and biological indicating that the site of PEG molecule attachment is one of the important factor to develop PEG-proteins as potential therapeutic agents.

• Applied Biological Chemistry
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• v.23 no.1
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• pp.23-30
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• 1980

Labelling of chromatin proteins with 32P was observed after incubating isolated liver nuclei with $[{\gamma}-32P]$ ATP for 5 minutes at $37^{\circ}C$. The pattern of labelling with 32P was examined on SDS polyacrylamide gel electrophoresis with nuclei from rats maintained in a starvation state for 48 hours, following refeeding for 12 hours; and from fed streptozotocin-diabetic rats with insulin injection 6 hours before sacrifice. With 48h starved rat liver nuclei the level of phosphorylation for 0.14M NaCl soluble proteins was decreased in the molecular weights between 41,000 and 200,000 daltons relative to normal controls. Refeeding the starved rats reversed the change of phosphorylation pattern over 12 hour The level of phosphorylation for five phenol soluble non-histone proteins with molecular weights above 59,000 daltons was somewhat decreased with 48h starved rat liver nuclei as compared with that of normal controls. Starvation also decreased the phosphorylation level of major histones in relation to normal controls. The experiment with insulin injection into fed streptozotocin-diabetic rats showed the tendency to increase phosphorylation of 0.14M NaCl soluble proteins (130,000 dalton protein) and phenol soluble non-histone proteins (155,000 dalton protein). The phosphorylation level of histones appeared to be invariant under the experimental conditoins employed here. These results suggest the possibility that the phosphorylation and dephosphorylation of 0.14M NaCl soluble proteins and $H_1$ histone precede those of other chromatin associated nuclear proteins, It is of interest to find that insulin signal was correlated to phosphorylation of nuclear proteins while glucagon signet dephosphorylated nuclear proteins.

• Journal of Periodontal and Implant Science
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• v.39 no.sup2
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• pp.231-237
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• 2009

Purpose: Cytolethal distending toxin (CDT) is a family of heat-labile cytotoxins produced by several gram-negative mucosa-associated pathogens, including Aggregatibacter actinomycetemcomitans. CDT is well known to be capable of inducing growth arrest, morphological alterations, and eventually death in various cells. CDT belongs to a tripartite $AB_2$ toxin (CdtB: the enzymatic A subunit; CdtA and CdtC: the heterodimeric B subunit). Previous studies proposed that CdtA and CdtC together bind to a cell surface receptor and glycolipids act as a receptor for A. actinomycetemcomitans CDT (AaCDT). In this study, recombinant CdtA and CdtC proteins of AaCDT were co-expressed in a bacterial expression system and tested for their affinity for $GM_1$ ganglioside. Methods: The genes for CdtA and CdtC from A. actinomycetemcomitans Y4 were utilized to construct the expression vectors, pRSET-cdtA and pET28a-cdtC. Both CdtA and CdtC proteins were expressed in Escherichia coli BL21(DE3) and then purified using hexahistidine (His6) tag. The identity of purified protein was confirmed by anti-His6 antibody and monoclonal anti-CdtA antibody. Furthermore, the affinity of recombinant protein to $GM_1$ ganglioside was checked through ELISA. Results: Recombinant CdtA and CdtC proteins were expressed as soluble proteins and reacted to anti-His6 and monoclonal anti-CdtA antibodies. ELISA revealed that purified soluble CdtA-CdtC protein bound to $GM_1$ ganglioside, while CdtA alone did not. Conclusions: Co-expression of CdtA and CdtC proteins enhanced the solubility of the proteins in E. coli, leading to convenient preparation of active CdtA-CdtC, a critical material for the study of AaCDT pathogenesis.