• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.663-669
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• 2000

The fur (ferric uptake regulation) expression vector pMON2064 was modified to produce a Fur-fusion expression vector. A kinker site, factor Xa cleavage site, and several restriction endonuclease sites were introduced to facilitate easy cloning and isolating of the fusion protein. The resulting fusion expression vector, pMONSTER, was then used to make fusion expression vector, pMONSTER, was then used to make fusion proteins with $\beta$-galactosidase and the protease of the human immunodeficiency virus type 1 (HIV-1 PR). Strain SW4020 harboring the Fur $\beta$-galactosidase fusion vector produced blue colonies on a 5-bromo-4-chloro-3-indolyl-$\beta$-D-galactoside plate and the resulting 133 kDa fusion protein reacted with an anti-Fur antibody. The strain harboring the Fur-HIV-1 PR fusion vector produced a 29 kDa fusion protein, which also reacted with an anti-Fur antibody. The Fur-HIV-1 PR fusion protein was purified by a single column application that was designed to isolate the Fur protein. The purified Fur-HIV-1 PR fusion protein digested with factor Xa cleaved a recombinant Gag protein to release smaller fragments, including a p24 capsid protein. The Fur-HIV-1 PR fusion protein itself did not exhibit any proteolytic activity.

• BMB Reports
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• 제31권5호
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• pp.427-431
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• 1998

In the previous report (Choi et al., 1997), the angiogenin binding peptides identified from a phage-peptide library were analyzed by using the fusion proteins composed of the Escherichia coli maltose binding protein and its corresponding peptides. However, it was difficult to obtain a sufficient amount of the fusion proteins required for further analysis because of the low expression level. We now report a high level expression of the fusion protein and analysis of its anti-angiogenin activity. The use of strong T7 promoter and removal of signal sequence allowed about a 20-fold increase in the expression efficiency of the fusion protein. We were able to obtain about 10 mg of purified fusion protein from one liter of culture. The purified fusion protein showed angiogenin-specific affinity and inhibited the binding of biotinylated actin to human angiogenin at $IC_{50}$ of 0.6 mM. Its anti-angiogenin activity was also revealed by the chorioallantoic membrane assay.

• BMB Reports
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• 제32권4호
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• pp.345-349
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• 1999

We fused the promoter region of an H3.2 chicken histone gene, whose expression is dependent on the cell cycle, to the 5' coding region of an H3.3 chicken histone gene, which is expressed constitutively at a low level throughout the cell cycle. This fusion gene showed a cell cycle-regulated pattern of expression, but in a different manner. The mRNA level of the fusion gene increase during the S phase of the cell cycle by about 3.7-fold at 6 h and 2.7-fold at 12 h after the serum stimulation. The mRNA level of the intact H3.2 gene, however, increased by an average of 3.6-fold at 6 h and 8.7-fold at 12 h. This different expression pattern might be due to the differences in their 3' end region that is responsible for mRNA stability. The 3' end of the H3.2 mRNA contains a stem-loop structure, instead of a poly(A) tail present in the H3.3 mRNA. We also constructed a similar fusion gene using a H3.3 histone gene whose introns had been eliminated to rule out the possibility of involvement of the introns in cell cycle-regulated expression. The expression of this fusion gene was almost identical to the fusion gene made previously. These results indicate that the promoter region of the H3.2 gene is only partially responsible for its expression during the S phase of the cell cycle.

• Archives of Pharmacal Research
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• 제19권4호
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• pp.275-279
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• 1996

To study the functional roles of dopamine receptors, we decided to raise antibodies against these proteins. To make antigen, we expressed the whole 3rd cytoplasmic loop of dopamine receptors in a fusion protein with glutathione-S-transferase (GST). $For D_2\; and\; D_3$ receptors, it was successful to express and purify fusion proteins for the whole 3rd cytoplasmic loops. However, we could not express the fusion protein for the whole 3rd cytoplasmic loop of $D_4$ dopamine receptor in the bacteria. To study the causes that prevent the bacterial expression of the GST-fusion protein of the 3rd cytoplasmic loop of $D_4$ dopamine receptor, we conducted more detailed studies on $D_4$ dopamine receptor. To locate the region which prevents bacterial expression, we made sequential constructs in the 3rd cytoplasmic loop decreasing the size step by step, and confirmed their expressions in the SDS PAGE. It was found that certain regions of 3rd cytoplasmic loop of $D_4$ dopamine receptor, located in N-terminal side of the 3rd cytoplasmic loop of $D_4$ dopamine receptor suppress the bacterial expression of fusion protein.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.65-70
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• 2002

A TGF-${\beta}1$/GFP monomeric fusion protein was cloned from pPK9A and pGFP-Cl plasmid by PCR amplification. The fusion protein was expressed in a $Bac-To-Bac^{TM}$ baculovirus expression system. A 45 kDa fusion protein was purified using an Ni-NTA column with 300 mM imidazol from a cell lysate infected with recombinant viruses for 72 h post-infection. The fusion protein cross-reacted with the commercial $TGF-{\beta}1$ polyclonal Ab as well as Ab raised against a precursor, monomeric $TGF-{\beta}1$, and GFP. The binding activity of the fusion protein with a $TGF-{\beta}1$ receptor was examined. Fluorescence was observed in Mv1Lu cells, yet not in insect cells treated with the fusion protein. No fluorescence was detected in Mv1Lu cells incubated with the fusion protein treated with Ab prior to the binding reaction, or with GFP alone, thereby indicating that the binding of the fusion protein was specific to $TGF-{\beta}1$ with a receptor.

• International Journal of Industrial Entomology and Biomaterials
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• 제8권2호
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• pp.145-149
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• 2004

The expression of a fusion protein comprised of the B. thuringiensis crystal protein, Cry1Ac, and enhanced green fluorescent protein (EGFP) in Escherichia coli XLl-blue was examined. Three recombinant plasmids were transformed into E. coli XL1-blue and named as ProAc/Ec, MuEGFP/Ec and ProMu-EGFP/Ec, respectively. All transformants were observed by light and fluorescence microscopy at mid-log phase. The expression in E. coli transformants, ProMu-EGFP/Ec and MuEGFP/Ec, exhibited bright enough fluorescence to be observed. Furthermore, ProMu-EGFP/Ec produced fluorescent inclusions, which may have been recombinant crystals between EGFP and Cry1Ac while MuEGFP/Ec expressed soluble EGFP in cell. In SDS-PAGE, ProAc/Ec had 130 kDa crystal protein band and MuEGFP/Ec had thick 27 kDa EGFP band. However, ProMu-EGFP/Ec had about 150 kDa fusion protein band. Accordingly, these results indicated that a fusion protein between the B. thuringiensis crystal protein and a foreign protein under the lacZ promoter was successfully expressed as granular structure in E. coli. It is suggested that the E. coli expression system by N-terminal fusion of B. thuringiensis crystal protein may be useful as excellent means for fusion expression and characterization of B. thuringiensis fusion crystal protein.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.728-732
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• 2007

In the present article, a novel fusion expression vector for Escherichia coli was developed based on the pTORG plasmid, a derivative of pET32a. This vector, named pT7MT(GenBank Accession No DQ504436), carries a T7 promoter and it drives the downstream gene encoding Metallothionein 2A(MT2A). There are in-framed multiple cloning sites(MCS) downstream of the MT2A gene. A target gene can be cloned into the MCS and fused to the C-terminal of the MT2A gene in a compatible open reading frame(ORF) to achieve fusion expression. The metal-binding capability of MT2A allows the purification of fusion proteins by metal chelating affinity chromatography, known as $Ni^{2+}$-affinity chromatography. Using this expression vector, we successfully got the stable and high-yield expression of MT2A-GST and MT2A-Troponin I fusion proteins. These two proteins were easily purified from the supernatant of cell lysates by one-step $Ni^{2+}$-affinity chromatography. The final yields of MT2A-GST and MT2A-Troponin I were 30mg/l and 28mg/l in LB culture, respectively. Taken together, our data suggest that pT7MT can be applied as a useful expression vector for stable and high-yield production of fusion proteins.

• 패션비즈니스
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• 제13권4호
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• pp.60-76
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• 2009

Expression of woman characters in fusion traditional dramas plays a role of making the progress of dramas not bigoted and new. Especially, woman characters have a high weight as a heroine by an increase in their image in fusion traditional dramas. Expression of characters harmonizing modern with tradition has also given a help in reflecting a trend of the present times. Hair style and face makeup of woman characters in fusion traditional dramas are in the process of fusionization getting an effect from postmodernism. They are expressing the hair style of symbolization modern elements of hair style to traditional hair styles. They also expressed a neutral image with faded hair styles in the shaggy cut style and dye of neoplaticism. Neo-hippie style was changed into the style of naturalism and nationalism and the hair style braided in various branches as the one of Indians was changed into a primitive and national feature. It is producing a new style by permitting even a long-hair permanent wave hair style. Expression of straight hair style, a long-hair shaggy & bulging wave style and a hair style of neoplaticism, was distinguished. In the side of face makeup, they expressed its luxurious and splendid style by attaching great importance to its luster and are exposing images of characters by a smoky makeup emphasizing eye lines. Their face makeup was almost never separated from present dramas as using pearl shadow in a glossy lips makeup and color, which made it possible to express it more dramatically in fusion traditional dramas than in present dramas. In the event of the makeup element of fusion traditional dramas permitting diversity, the character makeup of fusion traditional dramas made a foundation to show people diverse elements of makeup by mix & match a present elements and past elements of historical research, which made it possible to express a unique makeup or a special makeup. Diverse makeup expressions were limited by reflection of illumination even in the existing videos. Therefore, 'Fusion' made it possible to express it more freely in fusion traditional dramas than in present dramas.

• Bulletin of the Korean Chemical Society
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• 제28권9호
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• pp.1549-1552
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• 2007

The cytoplasmic domain of syndecan-4, a type I transmembrane heparan sulfate proteoglycan, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The cytoplasmic domain was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The integrity of the resulting peptide fragment was checked by MALDI-TOF and NMR spectroscopy. The yield of the peptide was 3.0-1.5 mg per liter in LB or minimal medium, respectively. The recombinant expression and purification of this domain will enable us its structural and functional studies using multidimensional NMR spectroscopy.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.550-553
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• 2003

$Hisx_6-GFPuv-Cyt$ c fusion protein을 E. coli 균주 JM109과 BL21 각각에서 발현시킨 결과 발현온도는 $30^{\circ}C$보다 $37^{\circ}C$에서, BL21보다 JM109에서의 발현량이 더 많았다. 그러나 JM109, $37^{\circ}C$에서 발현시킨 fusion protein의 $Ni^{2+}-IDA-agarose$ purification결과 약 45kDa 부근의 fusion protein의 density가 감소되었음을 SDS-PAGE analysis을 통해 알 수 있었다. 또한 western blotting analysis를 통해 이 impurity가 degraded fusion임을 확인 할 수 있었다. degraded fusion은 BL21 균주에서 발현시킨 fusion protein에서도 생성됨을 확인하였다. 모든 결과를 종합해 볼때 $Hisx_6-GFPuv-Cyt$ c fusion protein의 발현은 IM109, $37^{\circ}C$에서 더 많았지만, BL21, $37^{\circ}C$에서 expression시킨 fusion protein이 보다 안정하다고 판단 되어진다. 향후 fusion protein이 bioelectronic device에 적용되려면 degraded fusion protein의 생성을 줄여 activity를 유지하도록 안정한 형태로 발현되어 순수하게 분리정제 되어야 하겠다.