• 생명과학회지
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• 제30권11호
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• pp.931-938
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• 2020

TCTP는 다양한 진핵생물에서 풍부하게 존재하는 단백질 중에 하나이며, 암, 세포 증식, 유전자 조절 등과 관련된 세포의 생리학적 기작에서 중요한 역할을 담당하는 것으로 알려져 왔다. 더구나, TCTP는 dithiothreitol (DTT)나 hydrogen peroxide (H₂O₂)에 의해 유도되는 산화적 스트레스에 대한 저항성에 관여하는 중요한 단백질로 주목 받아 왔다. 한편, 극지역 서식 생물들은 강한 자외선에 의해 발생한 활성산소를 조절하기 위한 다양한 항산화 방어체계를 가지고 있다. 본 연구에서는 북극 동물플랑크톤 Calanus glacialis에서 분리된 TCTP가 산화적 스트레스 하에서 E. coli 세포의 저항성에 미치는 효과를 관찰하였다. C. glacialis에서 분리된 TCTP 유전자(ORF 522 bp) 서열을 분석하였고, 약 23 kDa의 재조합 단백질을 제작하였다. 관찰 결과, TCTP 재조합 단백질이 E. coli 세포에서 과발현 되었을 때, 세포들은 H₂O₂에 의해 유도된 산화적 스트레스에 대한 저항성이 증가하는 것을 확인하였다. 본 관찰을 통해, 북극 C. glacialis TCTP 단백질의 항산화 조절자로서의 역할에 대한 가능성을 처음으로 제시하였다.

• BMB Reports
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• 제46권7호
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• pp.358-363
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• 2013

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein.

• 미생물학회지
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• 제45권4호
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• pp.419-424
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• 2009

발광 세균 Photobacterium과 Vibrio의 lux 유전자가 삽입된 재조합 플라스미드를 유전자 전이시킨 발광표현형 대장균이 내는 빛의 세기를 조사하였다. 여러 대장균 균주에 형질전환 시켰는 바, 빛을 내는데 관여하는 효소들을 코드하는 유전자를 모두 포함하는 Photobacterium leiognathi lux 오페론이 삽입된 재조합플라스미드(PlXba.pT7-3)가 형질전환된 대장균 43R (Escherichia coli 43R) 균주에서는 발광세기가 다른 균주에 비해 1,000배 이상 되었으며, Vibrio harveyi luxA 유전자와 luxB 유전자를 융합시킨 유전자가 삽입된 재조합플라스미드(VhluxAB.pT7-5)가 형질전환 된 대장균 43R (E. coli 43R)에서는 기질인 fatty aldehyde를 가하면 단일 콜로니에서도 빛을 볼 수 있게 되는 대장균 형질전환체를 얻었다. 또한 이들이 중금속에 노출되었을 때 생물발광이 감소하였으며, 이들 발광 대장균 형질 전환체가 담긴 고정화된 세포에서도 빛을 내는 것을 관측함으로써 이들 실험 결과들이 lux 유전자를 활용한 바이오센서 시스템 개발의 기반 기술이 될 가능성을 보였다.

• 한국미생물·생명공학회지
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• 제29권1호
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• pp.18-24
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• 2001

누에 핵다각체병 바이러스의 gp64 프로모터를 이용한 transient 발현 벡터를 제작하기 위해서 gp64 유전자의 구조를 분석하였다. Southern blotting 분석을 통해 genome DNA에서 gp64 유전자를 탐색하기 gp64 구조유전자를 포함하는 2,277 nucleotide의 염기를 분석하였으며 gp64의 early, late 프로모터 발현을 조절하는 인자들을 확인하였다. gp64프로모터를 이용한 transient 발현 벡터를 제작하고 외래유전자로써 lacZ 유전자를 Bm5 세포주에서 transient 발현시켰다. 세포주 내에 도입된 플라스미드 DNA의 안정성을 확인하였으며, gp64 프로모터의 외래유전자 발현성 여부를 조사하기 위하여 gp64 프로모터 하에 laxZ 유전자를 가지는 재조합 바이러스를 제작하고 $\beta$-galactosidase in 냐셔 staining을 수행한 결과 전체적인 발현량은 매우 약한 것으로 판단되었다. BmNPV-K1의 gp64 프로모터를 이용한 벡터는 더욱 민감한 표지 유전자를 발현시켜 재조합 바이러스의 분리에 이용하거나 숙주세포에 독성을 보이는 유전자 산물의 소량 발현에 더욱 유용할 것으로 판단된다.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Animal cells and systems
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• 제11권2호
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• pp.175-182
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• 2007

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.

• IMMUNE NETWORK
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• 제24권1호
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• pp.1.1-1.6
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• 2024

IL-18 binding protein (IL-18BP) was originally discovered in 1999 while attempting to identify an IL-18 receptor ligand binding chain (also known as IL-18Rα) by subjecting concentrated human urine to an IL-18 ligand affinity column. The IL-18 ligand chromatography purified molecule was analyzed by protein microsequencing. The result revealed a novel 40 amino acid polypeptide. To isolate the complete open reading frame (ORF), various human and mouse cDNA libraries were screened using cDNA probe derived from the novel IL-18 affinity column bound molecule. The identified entire ORF gene was thought to be an IL-18Rα gene. However, IL-18BP has been proven to be a unique soluble antagonist that shares homology with a variety of viral proteins that are distinct from the IL-18Rα and IL-18Rβ chains. The IL-18BP cDNA was used to generate recombinant IL-18BP (rIL-18BP), which was indispensable for characterizing the role of IL-18BP in vitro and in vivo. Mammalian cell lines were used to produce rIL-18BP due to its glycosylation-dependent activity of IL-18BP (approximately 20 kDa). Various forms of rIL-18BP, intact, C-terminal his-tag, and Fc fusion proteins were produced for in vitro and in vivo experiments. Data showed potent neutralization of IL-18 activity, which seems promising for clinical application in immune diseases involving IL-18. However, it was a long journey from discovery to clinical use although there have been various clinical trials since IL-18BP was discovered in 1999. This review primarily covers the discovery of IL-18BP along with how basic research influences the clinical development of IL-18BP.

• IMMUNE NETWORK
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• 제21권3호
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• pp.23.1-23.16
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• 2021

Chemokines are key factors that influence the migration and maintenance of relevant immune cells into an infected tissue or a tumor microenvironment. Therefore, it is believed that the controlled administration of chemokines in the tumor microenvironment may be an effective immunotherapy against cancer. Previous studies have shown that CCL3, also known as macrophage inflammatory protein 1-alpha, facilitates the recruitment of dendritic cells (DCs) for the presentation of tumor Ags and promotes T cell activation. Here, we investigated the role of CCL3 in regulating the tumor microenvironment using a syngeneic mouse tumor model. We observed that MC38 tumors overexpressing CCL3 (CCL3-OE) showed rapid regression compared with the wild type MC38 tumors. Additionally, these CCL3-OE tumors showed an increase in the proliferative and functional tumor-infiltrating T cells. Furthermore, PD-1 immune checkpoint blockade accelerated tumor regression in the CCL3-OE tumor microenvironment. Next, we generated a modified CCL3 protein for pre-clinical use by fusing recombinant CCL3 (rCCL3) with a non-cytolytic hybrid Fc (HyFc). Administering a controlled dose of rCCL3-HyFc via subcutaneous injections near tumors was effective in tumor regression and improved survival along with activated myeloid cells and augmented T cell responses. Furthermore, combination therapy of rCCL3-HyFc with PD-1 blockade exhibited prominent effect to tumor regression. Collectively, our findings demonstrate that appropriate concentrations of CCL3 in the tumor microenvironment would be an effective adjuvant to promote anti-tumor immune responses, and suggest that administering a long-lasting form of CCL3 in combination with PD-1 blockers can have clinical applications in cancer immunotherapy.

• KSBB Journal
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• 제23권3호
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• pp.205-212
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• 2008

본 연구의 목적은 위치특이적 펩타이드 및 단백질을 사용하여 재조합 대장균에서 활성형 인간 상피세포성장인자(hEGF)를 고효율로 발현할 수 있는 방법을 찾아내는 데 있다. 재조합 대장균내 cytoplasm 및 periplasm 영역에서 hEGF의 발현을 위해 각각 세개의 응합 펩타이드 및 단백질을 선정하여 상호 비교하였다. 재조합 대장균에서 hEGF의 발현유도시 대부분 불용성 단백질로 생산되는 현상을 극복하기 위해 cytoplasm영역에서는 ATS, thioredoxin, 리파제를 융합파트너로 사용하였으며 periplasm 영역에서는 foldase인 DsbA와 DsbC, 용융성 고발현 단백질인 maltose binding protein을 선택하여 사용하였다. Periplasm영역에서 발현유도를 시키는 융합단백질의 경우 cytoplasm영역에서의 발현양도 용융성 형태로 고발현 되는 것을 알 수 있었으며 전체적으로 약 2배가량의 용융성 형태로 발현되었다. hECF의 발현율을 가장 높일 수 있는 융합단백 질은 maltose binding protein이었으나 발현된 융합단백질의 24%가 불용성 단백질로 형성되어 활성형 형태로 얻는 데 한계가 있었으며, 활성형 형태로 hEGF의 발현을 위해서는 DsbA를 응합단백질로 사용한 경우에 18.1 mg/L로 가장 높은 발현농도를 보였다. Cytoplasm 영역에서 발현유도를 한 경우에는 ATS와 thioredoxin을 응합파트너로 hEGF를 발현한 경우 용융성 형태로 높은 발현율을 보였다. 특히 ATS와 같은 펩타이드를 N-말단에 융합시킨 경우 불용성을 방지하는 효과를 보여 이황화결합의 불완전성이나 소수성으로 인해 불용성 단백질로 발현되는 기존의 단백질을 활성형 형태로 발현하는데 될 수 있음을 확인할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1232-1238
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• 2004

To investigate the production and characteristics of thermostable levan sucrase from Rahnella aquatilis ATCC 33071, the levan sucrase gene from R. aquatilis was cloned and expressed in Escherichia coli without induction system. Expression of levansucrase gene in E. coli had no notable or detrimental effect on the growth of host strain, and the recombinant levan sucrase exhibited levan synthesis activity. Levansucrase was secreted to the periplasm in E. coli, and addition of $0.5\%$ glycine yielded further secretion of levansucrase to the growth medium and resulted in an increase of total levansucrase activity. Furthermore, the cellular levansucrase was evaluated for the production of levan by using toluene­permeabilized whole-cells. The levansucrase was thermostable at $37^{\circ}C$. The molecular size oflevan was $1{\times}\;10^{6}$ Da, as determined by HPLC, and the degree of polymerization of levan varied with incubation temperatures: Low incubation temperature was preferable for the production of high-molecular size levan. The present study demonstrated that the mass production of levan and levan oligosaccharides can be achieved by glycine supplementation to the growth medium or by toluene­permeabilized whole-cells.