• 한국수정란이식학회지
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• 제14권1호
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• pp.1-8
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• 1999

The efficiency of transgenic livestock animal production may be improved by early selection of transgenci preimplantation embryos. To examine the possibility of GFP gene as a non-invasive marker for the early screening of transgenic embryo, the GFP gene was microinjected into rabbit zygotes and the later stages of preimplantation embryos were examined for the expression of GFP. The presence of injected DNA was detected by PCR analysis and the expression of GFP was detected by observing green fluorescence in embryos under a fluorescent microscope. Out of 108 GFP gene-injected rabbit zygotes, seventy three(67.6%) were fluorescence-positive. When 11 fluroresecence-positive blastocysts were analyzed for the presence of GFP gene by PCR, 6(54.5%) were positive, and all of the 8 flrouescence-negative blastocysts were also negative by PCR. The results indicate that the screening of transgene in rabbit embryos by PCR analysis and GFP detection could be a promising method for the preselection of transgenic embryos.

• 한국정보통신학회논문지
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• 제18권8호
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• pp.2017-2022
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• 2014

폴리히드론 프로모터, 수포성구내염 바이러스G, 폴리A, 사이토메가바이러스 프로모터, 강화녹색형광단백질, 단백질전달부위 유전자 등을 포함한 새로운 베큘로바이러스 시스템이 제조되었다. 본 재조합벡터 시스템은 인간 섬유아세포에 적용하여 시험하였고 재조합된 유전자의 전달과 유전자 발현을 대조 벡터시스템과 비교하였다. 본 연구로부터 새롭게 제작된 본 베큘로바이러스 시스템이 유전자의 전이와 유전자 발현 면에서 대조 벡터시스템 보다 고효율을 나타내었다.

• 생명과학회지
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• 제28권12호
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• pp.1416-1423
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• 2018

Biotin에 강한 결합 친화력($K_D=10^{-14}M$)과 함께 streptavidin의 tetramer 특징은 VHH 항체를 streptavidin에 융합시키게 하여 biotinylated horseradish peroxidase를 사용하는ELISA 와Western blot analysis 등의 면역분석법에서 VHH 항체의 항원결합력을 증가시키는데 응용 가능하다. 이를 응용하기 위해 우리는 Streptomyces avidinii 염색체 DNA로부터 PCR을 통해 streptavidin유전자를 증폭하고 이를 green fluorescent protein항원에 특이적으로 결합하는 8B9 VHH 항체유전자에 융합시켰다. 대장균에서 수용성 융합단백질로 발현시키기 위해 pUC119 플라스미드에 기초한 발현시스템을 사용하였다. 즉 lacZ promoter를 사용하여 IPTG에 의해 단백질발현을 유도하게 하였으며, 아미노말단에 pelB leader를 두어 발현된 단백질의 periplasmic space로 이동하게 하여 수용성 단백질형태의 분비를 촉진하였으며 카르복시말단에 6개의 polyhistidine tags를 두어 $Ni^+$-NTA-agarose column을 사용하여 발현된 단백질을 정제하였다. Streptavidin이 biotin에 강하게 결합함으로 대장균에 독성을 나타냄에도 불구하고 본 수용성 융합단백질은 성공적으로 발현되었다. SDS-PAGE에서 가열하는 경우 변성되어 30.6 kDa를, 가열하지 않는 경우에는 자연 상태의 122.4 kDa을 나타내었다. 이는 8B9 VHH항체에 융합된 streptavidin moiety에 의해 monomer subunit가 비공유결합으로 tetramerization됨을 제시해준다. 또한 본 융합단백질은 ELISA와 Westernblot analysis에서 보여진 것처럼 parental streptavidin과 유사한 biotin결합력과 green fluorescent protein항원 결합력을 모두 나타내었다. 결론적으로 streptavidin에 융합된 8B9 VHH 항체형태의 융합단백질은 대장균에서 수용성 tetramer로 성공적으로 발현 및 정제되었으며 biotin과 green fluorescent protein 항원에 동시에 결합함으로써 tetrameric and bifunctional VHH 항체제조의 가능성을 제시해주었다.

• Journal of Microbiology
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• 제42권4호
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• pp.340-345
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• 2004

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.797-804
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• 2000

The human immunodeficiency virus type 1 (HIV-1) Tat is one of the viral gene products essential for HIV replication. The exogenous Tat protein is transduced through the plasma membrane and then accumulated in a cell. The basic domain of the Tat protein, which is rich in arginine and lysine residues and called the protein transduction domain (PTD), has been identified to be responsible for this transduction activity. To better understand the nature of the transduction mediated by this highly basic domain of HIV-1 Tat, the Green Fluorescent Protein (GFP) was expressed and purified as a fusion protein with a peptide derived from the HIV-1 Tat basic domain in Escherichia coli. The transduction of Tat-GFP into mammalian cells was then determined by a Western blot analysis and fluorescence microscopy. The cells treated with Tat-GFP exhibited dose- and time-dependent increases in their intracellular level of the protein. the effective transduction of denatured Tat-GFP into both the nucleus and the cytoplasm of mammalian cells was also demonstrated, thereby indicating that the unfolding of the transduced protein is required for efficient transduction. Accordingly, the availability of recombinant Tat-GFP can facilitate the simple and specific identification of the protein transduction mediated by the HIV-1 Tat basic domain in living cells either by fluorescence microscopy or by a fluorescence-activated cell sorter analysis.

• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.152-159
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• 2014

The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Co-expression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and $PDGFR{\beta}$ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and $PDGFR{\beta}$ fusion proteins were purified by $Ni^{2+}$ affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and $PDGFR{\beta}$ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics.

• Journal of Microbiology and Biotechnology
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• 제27권7호
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• pp.1345-1358
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• 2017

The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N-acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

• Bulletin of the Korean Chemical Society
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• 제25권3호
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• pp.345-346
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• 2004

• Bulletin of the Korean Chemical Society
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• 제34권1호
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• pp.29-30
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• 2013

• Journal of Microbiology and Biotechnology
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• 제10권2호
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• pp.264-266
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• 2000

A new GFP-based T-vector for cloning of PCR products was developed by using a green fluorescent protein (GFP) as a mafker. In order to facilitate the DNA inserts, multiple restriction sites, SP6 and T7 RNA polymerase promoter sites, were introduced close to the PCR DNA insertion site of a pCRGv vector. The XcmI-digested pHNT plasmid can be used to clone a 3' A-overhanged PCR DNA amplified by Taq DNA polymerase. A potential method of easing some difficulties from its use along with its cost savings proveded by this vector are likely to lead to the replacement of other T-vectors for PCR DNA cloning.