• 한국미생물·생명공학회지
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• 제43권3호
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• pp.212-218
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• 2015

Fucosyltransferase는 퓨코실화된 올리고당을 생성하는데 필수적인 효소로서, GDP-β-L-fucose로 부터 fucose를 수용체로 전이시켜 알파 글리코사이드 결합을 형성하는 과정을 촉매한다. 하지만 Escherichia coli 에서 발현시켰을 때, 대부분의 경우 inclusion body를 형성하여 비활성으로 생성되었다. 따라서 본 연구에서는 Helicobacter pylori 26695에서 유래한 α1,2-fucosyltransferase (FucT2) 유전자의 수용성 발현을 위하여, E. coli에서 샤페론 단백질인 GroEL, GroES, DnaK, DnaJ, GrpE과 함께 동시발현시켰다. SDS-PAGE 분석결과, 5가지 샤페론 단백질과 함께 발현되었으며, 수용성 FucT2 단백질이 증가하였고 효소활성은 5배 증가하였다. 결론적으로, 샤페론 동시발현기술을 이용하여 대장균에서 FucT2 수용성 생산을 증가시킬 수 있었으며 본 수용성 효소는 퓨코실화된 올리고당을 효율적으로 생산하는데 이용될 수 있다.

• Journal of Microbiology and Biotechnology
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• 제32권11호
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• pp.1471-1478
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• 2022

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (Kan^R) should be dependent on FucT2 solubility. Kan^R was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1092-1097
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• 2009

A novel exopolysaccharide named Ebosin was produced by Streptomyces sp. 139, with medicinal activity. Its biosynthesis gene cluster (ste) has been previously identified. For the functional study of the ste7 gene in Ebosin biosynthesis, it was disrupted with a double crossover via homologous recombination. The monosaccharide composition of EPS-7m produced by the mutant strain Streptomyces sp. 139 ($ste7^-$) was found altered from that of Ebosin, with fucose decreasing remarkably. For biochemical characterization of Ste7, the ste7 gene was cloned and expressed in Escherichia coli BL21. With a continuous coupled spectrophotometric assay, Ste7 was demonstrated to have the ability of catalyzing the transfer of fucose specifically from GDP-$\beta$-L-fucose to a fucose acceptor, the lipid carrier located in the cytoplasmic membrane of Streptomyces sp. 139 ($ste7^-$). Therefore, the ste7 gene has been identified to code for a fucosyltransferase, which plays an essential role in the formation of repeating sugars units during Ebosin biosynthesis.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1657-1662
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• 2012

Fucosyltransferase IV (FUT4) has been implicated in cell adhesion, motility, and tumor progression in human epidermoid carcinoma A431 cells. We previously reported that it promotes cell proliferation through the ERK/MAPK and PI3K/Akt signaling pathways; however, the molecular mechanisms underlying FUT4-induced cell invasion remain unknown. In this study we determined the effect of FUT4 on expression of matrix metalloproteinase (MMP)-12 induced by EGF in A431 cells. Treatment with EGF resulted in an alteration of cell morphology and induced an increase in the expression of MMP-12. EGF induced nuclear translocation of nuclear factor kB (NF-${\kappa}B$) and resulted in phosphorylation of $IkB{\alpha}$ in a time-dependent manner. In addition, ERK1/2 and p38 MAPK were shown to play a crucial role in mediating EGF-induced NF-${\kappa}B$ translocation and phosphorylation of $I{\kappa}B{\alpha}$ when treated with the MAPK inhibitors, PD98059 and SB203580, which resulted in increased MMP-12 expression. Importantly, we showed that FUT4 up-regulated EGF-induced MMP-12 expression by promoting the phosphorylation of ERK1/2 and p38 MAPK, thereby inducing phosphorylation/degradation of $I{\kappa}B{\alpha}$, NF-${\kappa}B$ activation. Base on our data, we propose that FUT4 up-regulates expression of MMP-12 via a MAPK-NF-${\kappa}B$-dependent mechanism.

• Reproductive and Developmental Biology
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• 제39권3호
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• pp.59-67
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• 2015

동물의 장기를 인간에게 이식하게 되면 초급성거부반응(Hyperacute rejection, HAR)이 일어난다. 초급성거부반응은 면역계의 구성요소 중 보체(complement)에 의해 일어나는 거부반응으로 돼지의 혈관세포 표면에 있는 $Gal{\alpha}$(1,3)Gal 당분자에 인간의 항체가 즉각 반응하기 때문에 일어나며, ${\alpha}1,3$-galactosyltransferase(${\alpha}1,3$-GT) 유전자는 돼지 혈관세포 표면의 $Gal{\alpha}$(1,3)Gal 당분자 생성에 관여한다. 따라서 인간에게 돼지의 장기를 이식하기 위해서는 ${\alpha}1,3$-galactosyltransferase 유전자를 제거하는 것이 필요한 것으로 알려져 있다. 본 연구실의 이전 연구에서, 시카고 미니돼지 귀체세포에서 상동 재조합(Homologous recombination)을 통해 ${\alpha}1,3$-galactosyltransferase 유전자가 제거된 체세포를 개발한 바 있으며, 이 체세포를 통하여 ${\alpha}1,3$-GT 유전자가 제거된 돼지도 생산된 바 있다. 본 연구에서는, human serum 처리 시 돼지 세포를 보호해 준다고 보고되고 있는 human complement regulator인 human Decay-accelerating factor(hDAF)와 human ${\alpha}1,2$-fucosyltransferase(hHT)유전자를 ${\alpha}1,3$-GT 유전자 위치에 gene targeting하여 동시에 hDAF와 hHT가 발현하는 체세포를 개발하였다. Knock-in vector는 hDAF와 hHT 두 유전자가 발현할 수 있도록 IRES로 연결하였으며, ${\alpha}1,3$-GT 유전자의 start codon을 이용하여 발현할 수 있도록 구축하였다. 구축한 vector는 electroporation을 통해 미니 돼지 체세포에 도입하였으며, PCR 결과, ${\alpha}1,3$-GT 유전자 위치에서 상동 재조합이 일어났음을 확인하였다. Positive-negative 선별 방법을 통해 얻은 gene targeting 된 체세포는 RT-PCR에 의해 hDAF와 hHT 유전자의 발현이 확인되었으며, 대조군(NIH minipig)에 비해 ${\alpha}1,3$-GT 유전자의 발현이 감소하였다. 또한 이들 세포에 100% human complement serum을 처리하였을 때 knock-in 세포가 대조군에 비해 30% 정도 더 높은 생존율을 보였다. 따라서 개발된 체세포는 이종간 장기이식을 위한 돼지 생산과 함께 이를 이용한 이종간의 장기 이식 시 초급성 거부반응을 억제하는 데 사용될 수 있을 것으로 생각된다.

• BMB Reports
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• 제44권12호
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• pp.772-781
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• 2011

Branched N-glycans are produced by a series of glycosyltransferases including N-acetylglucosaminyltransferases and fucosyltransferases and their corresponding genes. Glycans on specific glycoproteins, which are attached via the action of glycosyltransferases, play key roles in cell adhesion and signaling. Examples of this are adhesion molecules or signaling molecules such as integrin and E-cadherin, as well as membrane receptors such as the EGF and TGF-${\beta}$ receptors. These molecules also play pivotal roles in the underlying mechanism of a variety of disease such as cancer metastasis, diabetes, and chronic obstructive pulmonary disease (COPD). Alterations in the structures of branched N-glycans are also hall marks and are useful for cancer biomarkers and therapeutics against cancer. This mini-review describes some of our recent studies on a functional glycomics approach to the study of branched N-glycans produced by N-acetylglucosaminyltransferases III, IV, V and IX (Vb) (GnT-III, GnT-IV, V and IX (Vb)) and fucosyltransferase 8 (Fut8) and their pathophysiological significance, with emphasis on the importance of a systems glycobiology approach as a future perspective for glycobiology.

• 한국수정란이식학회지
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• 제31권3호
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• pp.199-205
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• 2016

The aim of this study was to investigate the effect of uterine histotroph on embryo development and the expression of cysteine-rich protein 2 (CRP2), coatomer subunit gamma-2 (G2COP), myoglobin (MYG), vascular endothelial growth factor D (VEGFD), collagen alpha 4 chain (COL4) and galactoside 3-L-fucosyltransferase 4 (FUT4) proteins in porcine embryo during pre-implantation. Uterine histotroph (UH) was collected from uterine horn on corpus albican phase, and embryos were cultured in porcine zygote medium with UH for 168 hours. Cleavage and blastocyst formation of embryo were detected at 168 hours after in vitro fertilization. And CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were observed using confocal laser microscope. In results, embryo cleavage rate was not significantly changed by UH, but blastocyst rate was significantly (P<0.05) decreased in UH-treated embryos. Moreover, CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were expressed in blastomere. CRP2 in embryo was significantly overexpressed (P<0.05), but not G2COP, MYG, VEGFD, COL4 and FUT4 proteins. In summary, UH on corpus albican phase was increased CRP2 protein in embryo, and inhibited blastocyst formation in preimplantation porcine embryos, suggesting that CRP2 may play an interrupter on embryo development in pigs.