• 산업기술연구
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• 제30권B호
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• pp.69-74
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• 2010

Chitinolytic activity was enhanced by coexpression of endo-chitinase gene (chiA) and chitobiosidase gene (chiB) from Serratia marcescens KFRI314 using constitutive expression vector, pHCEIA, in E. coli. Coexpression vector was constructed by inserting ribosome binding site (RBS) into junction between two chitinase genes. SDS-PAGE analyses showed that two chitinase were constitutively expressed while E. coli clones expressing two chitinases simultaneously increased halo size on colloidal chitin plate. Furthermore, the chitinolytic activities were much enhanced in coexpressed clones when degradation patterns of substrate analogues such as 4-MU-(NAG), $4-MU-(NAG)_2$,$4-MU-(NAG)_3$ were used. Consequently, the combined use of endochitinase and chitobiosidase greatly increased overall chitinolytic activities on recombinant E. coli clones.

• BMB Reports
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• 제43권9호
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• pp.609-613
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• 2010

The Dvl and Axin proteins, which are involved in the Wnt signaling pathway, each contain a conserved DIX domain in their sequences. The DIX domain mediates interaction between Dvl and Axin, which together play an important role in signal transduction. However, the extremely low production of DIX domain fragments in E. coli has prevented more widespread functional and structural studies. In this study, we demonstrate that the DIX domains of Dvl and Axin are expressed noticeably in a multi-cistronic system but not in a mono-cistronic system. Formation of the $DIX_{Dvl1}-DIX_{Axin1}$ complex was investigated by affinity chromatography, SEC and crystallization studies. Unstable DIX domains were stabilized by complexing with counterpart DIX domains. The results of the preliminary crystallization and diffraction of the $DIX_{Dvl1}-DIX_{Axin1}$ complex may prove useful for further crystallographic studies.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.308-311
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• 2006

In an attempt to increase production of LK8, an 86-amino-acid kringle fragment of human apolipoprotein(a) with three disulfide linkages, protein disulfide isomerase (PDI) was coexpressed in recombinant Saccharomyces cerevisiae harboring the LK8 gene in the chromosome. Whereas overexpression of the LK8 gene without coexpressing PDI was detrimental to both host cell growth and LK8 production, coexpression of PDI increased the LK8 production level by 2.5-fold in batch cultivation and 5.0-fold in fed-batch cultivation compared with the control strain carrying only the genomic PDI gene.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.601-602
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• 2003

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1350-1355
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• 2004

The sprA and sprB genes, encoding the chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB), and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from S. griseus and were overexpressed in various strains of S. griseus. When the sprT gene was introduced into S. griseus, trypsin activity increased 2-fold in the A-factor deficient mutant strain, S. griseus HH1, and increased 4-fold in the wild strain, S. grise us IFO 13350. However, there was no detectable increase of chymotrypsin activity in the transformants of S. griseus with either sprA or sprB, in contrast to the results obtained from S. lividans as a heterologous host. To solve the negative gene dosage effects in S. griseus, either the sprA or the sprB genes with their own ribosome binding sites were linked to the downstream of the entire sprT gene, and the coexpression efficiency was examined in S. lividans and S. griseus. The transformants of S. lividans with either pWHM3-TA (sprT+sprA) or pWHM3­TB (sprT+sprB) showed 3-fold increase of trypsin activity over that of the control, however, only the transformant of pWHM3-TB demonstrated 7-fold increase in chymotrypsin activity, indicating that the pWHM3-TB has a successful construction for the overexpression of chymotrypsin in Streptomyces. When the coexpression vectors were introduced into S. griseus IFO 13350, the trypsin level sharply increased by more than 4-fold, however, the chymotrypsin level did not increase. These results strongly suggest that the overexpression of the sprA and sprB genes is tightly regulated in S. griseus.

• 한국미생물·생명공학회지
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• 제44권1호
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• pp.55-61
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• 2016

본 연구에서는 $BH_4$를 대체할 수 있는 유용물질인 세피아프테린의 생산성 증대를 위하여 GTP 생합성 경로의 유전자들을 동시에 발현할 수 있는 재조합 대장균을 제작하였다. 세피아프테린을 생산할 수 있는 재조합 대장균에서 gmk, ndk 및 guaA-guaB 유전자를 동시에 발현함으로써 세포 내 GTP의 농도가 대조구에 비해 약 200% 이상 증가하였고 $126.1{\pm}9.3mg/l$의 세피아프테린이 생산되었는데 이 결과는 대조구보다 세피아프테린의 생산량이 약 43% 증가된 것이다. GTP 생합성에 관여하는 개별 유전자의 단독 발현 또는 두 가지 유전자의 동시 발현은 세포 내 GTP 농도 향상 큰 영향을 미치지 못했지만 네 가지 유전자 모두를 동시에 발현하는 경우는 세포 내 GTP 농도를 유의적으로 증가시킨다는 것이 확인되었다. 결론적으로 세포 내 GTP 생합성에 관여하는 guaA-guaB, gmk 및 ndk 유전자를 동시에 발현함으로써 재조합 대장균에서 세피아프테린의 생산성 증가를 달성하였다.

• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1945-1952
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• 2008

Sialylation, the attachment of sialic acid residues to a protein, can affect the biological activity and in vivo circulatory half-life of glycoproteins. Human ${\alpha}2$,3-sialyltransferase (${\alpha}2$,3-ST) and ${\beta}1$,4-galactosyltransferase (${\beta}1$,4-GT) are responsible for terminal sialylation and galactosylation, respectively. Enhanced sialylation of human erythropoietin (EPO) by the expression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT was achieved using recombinant Chinese hamster ovary (CHO) cells (EC1). The sialic acid content and sialylation of N-glycans were evaluated by HPLC. When ${\alpha}2$,3-ST was expressed in CHO cells (EC1-ST2), the sialic acid content (moles of sialic acid/mole of EPO) increased from 6.7 to 7.5. In addition, the amount of trisialylated glycans increased from 17.3% to 26.1 %. When ${\alpha}2$,3-ST and ${\beta}1$,4-GT were coexpressed in CHO cells (EC1-GTST15), the degree of sialylation was greater than that in EC1-ST2 cells. In the case of EC1-GTST15 cells, the sialic acid content increased to 8.2 and the proportion of trisialylated glycans was markedly increased from 17.3% to 35.5%. Interestingly, the amount of asialoglycans decreased only in the case of GTST15 cells (21.4% to 14.2%). These results show that coexpression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT is more effective than the expression of ${\alpha}2$,3-ST alone. Coexpression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT did not affect CHO cell growth and metabolism or EPO production. Thus, coexpression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT may be beneficial for producing therapeutic glycoproteins with enhanced sialylation in CHO cells.

• 원예과학기술지
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• 제32권5호
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• pp.684-693
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• 2014

저온, 건조, 염과 같은 비생물적 스트레스는 식물의 생리적 형태적 변화와 수확량 감소를 초래한다. 이러한 이유로 식물체는 불리한 환경을 극복하기 위해 다양한 대사과정에 관련된 유전자들간의 복잡한 상호 관계를 조절함으로써 저항성을 획득한다. 본 연구는 배추에서 염 스트레스에 반응하는 유전자를 다각적으로 분석하기 위해 상호발현 네트워크를 구축하였다. 네트워크를 구축하기 위하여 배추를 염스트레스 조건 하에서 시간 경과에 따라 KBGP-24K 마이크로어레이 분석을 실시한 [BrEMD (Brassica rapa EST and Microarray Database)] 실험 결과를 수집하여 분석하였다. 구축된 네트워크 모델은 1,853개 node, 5,740개 edge, 및 142개 connected component(상관계수 > 0.85)로 구성되었다. 구축된 네트워크 분석 결과, ROS 신호 전달을 통한 N$Na^+$ 수송활성화와 proline 축적이 배추의 염 저항성 획득과 밀접한 연관이 있는 것으로 판단하였다.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.2076-2080
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• 2007

The endoxylanase (GenBank Access No. U51675) of Bacillus spp. and endoglucanase (GenBank Access No. AY466436) of Trichoderma spp. were separately inserted downstream of the yeast constitutive ADHI promoter, resulting in three different plasmids (pAGX1, pAGX2, and pAGX3) according to the transcription direction of two genes. When the yeast transformants, S. cerevisiae SEY2102 harboring each expression plasmid, were grown on YPD medium, the total activities of the enzymes were approximately 3.01 unit/ml, 3.24 unit/ml, and 7.56 unit/ml for endoxylanase and 0.60 unit/ml, 0.54 unit/ml, and 0.39 unit/ ml for endoglucanase, in the following order: the pAGX1, pAGX2, and pAGX3. More than 70% of the endoxylanase and endoglucanase activities was found in the extracellular media.

• KSBB Journal
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• 제26권3호
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• pp.189-192
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• 2011

Much interest has been recently focused on the production of large quantities of hydrogen, due to its potential importance in our economy and needs in the petroleum and chemical industries. Formate dehydrogenase H (FDH-H) from Escherichia coli containing selenocysteine that oxidizes formate to carbon dioxide with the release of hydrogen is a component of the anaerobic formate hydrogen-lyase complex of E. coli. To make full use of FDH-H, we need effective expression condition. In this approach, we investigated the effect of pH on FDH-H stability and observed the effect of selenite and formate concentration on the activity of FDH-H. Additionally, coexpression of selenocysteine insertion genes were tried to improve the expression of FDH-H. The highest level of FDH-H expression was achieved by coexpression of selenocysteine insertion genes (pSUABC) as well as by the addition of $10\;{\mu}M$ selenite and 10 mM formate. At this optimized condition, a 2.6 fold elevation of expression of FDH-H was achieved.