• Title/Summary/Keyword: coexpression

Search Result 79, Processing Time 0.027 seconds

Enhancement of chitinolytic activity of by co-expression of endochitinase and chitobiosidase genes (Endochitinase와 Chitobiosidase 유전자의 동시발현에 의한 키틴분해 활성의 증가)

  • Kim, Jungtae;Choi, Shin-Geon
    • Journal of Industrial Technology
    • /
    • v.30 no.B
    • /
    • pp.69-74
    • /
    • 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.

  • PDF

Coexpression and protein-protein complexing of DIX domains of human Dvl1 and Axin1 protein

  • Choi, Seung-Hye;Choi, Kyung-Mi;Ahn, Hyung-Jun
    • BMB Reports
    • /
    • v.43 no.9
    • /
    • pp.609-613
    • /
    • 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.

Coexpression of Protein Disulfide Isomerase (PDI) Enhances Production of Kringle Fragment of Human Apolipoprotein(a) in Recombinant Saccharomyces cerevisiae

  • Cha Kwang-Hyun;Kim Myoung-Dong;Lee Tae-Hee;Lim Hyung-Kweon;Jung Kyung-Hwan;Seo Jin-Ho
    • Journal of Microbiology and Biotechnology
    • /
    • v.16 no.2
    • /
    • pp.308-311
    • /
    • 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.

Overexpression of sprA and sprB Genes is Tightly Regulated in Streptomyces griseus

  • KIM , YOON-HEE;CHOI, SI-SUN;KANG, DAE-KYUNG;KANG, SANG-SOON;JEONG, BYEONG-CHUL;HONG, SOON-KWANG
    • Journal of Microbiology and Biotechnology
    • /
    • v.14 no.6
    • /
    • pp.1350-1355
    • /
    • 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.

Enhancement of Sepiapterin Production in Recombinant Escherichia coli by Coexpression of the Genes for Guanosine Triphosphate(GTP) Biosynthesis (Guanosine triphosphate(GTP) 생합성 유전자의 동시 발현을 통한 재조합 대장균에서 세피아프테린의 생산 증대)

  • Park, Eun-Hee;Lee, Won-Heong;Kim, Myoung-Dong
    • Microbiology and Biotechnology Letters
    • /
    • v.44 no.1
    • /
    • pp.55-61
    • /
    • 2016
  • Sepiapterin, a precursor for tetrahydrobiopterin, is produced in higher mammals using guanosine triphosphate (GTP) as a biosynthetic intermediate. Four genes involved in GTP biosynthesis, namely those of guanosine monophosphate kinase (gmk), nucleoside diphosphate kinase (ndk), guanosine phosphate synthetase (guaA), and inosine-5'-monophosphate dehydrogenase (guaB), were expressed in sepiapterin-producing recombinant Escherichia coli BL21(DE3) to increase intracellular GTP concentration and to improve sepiapterin production concomitantly. Coexpression of gmk, ndk, guaA, and guaB, doubled the intracellular GTP concentration and increased the maximum sepiapterin concentration up to $126.1{\pm}19.3mg/l$ (an increase of 43% compared with control cells) in batch-cultivated recombinant E. coli.

Enhanced Sialylation of Recombinant Erythropoietin in CHO Cells by Human Glycosyltransferase Expression

  • Jeong, Yeon-Tae;Choi, One;Lim, Hye-Rim;Son, Young-Dok;Kim, Hong-Jin;Kim, Jung-Hoe
    • Journal of Microbiology and Biotechnology
    • /
    • v.18 no.12
    • /
    • pp.1945-1952
    • /
    • 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.

Construction of a Network Model to Reveal Genes Related to Salt Tolerance in Chinese Cabbage (배추 염 저항성 관련 유전자의 네트워크 모델 구축)

  • Lee, Gi-Ho;Yu, Jae-Gyeong;Park, Ji-Hyun;Park, Young-Doo
    • Horticultural Science & Technology
    • /
    • v.32 no.5
    • /
    • pp.684-693
    • /
    • 2014
  • Abiotic stress conditions such as cold, drought, and salinity trigger physiological and morphological changes and yield loss in plants. Hence, plants adapt to adverse environments by developing tolerance through complex regulation of genes related to various metabolic processes. This study was conducted to construct a coexpression network for multidirectional analysis of salt-stress response genes in Brassica rapa (Chinese cabbage). To construct the coexpression network, we collected KBGP-24K microarray data from the B. rapa EST and microarray database (BrEMD) and performed time-based expression analyses of B. rapa plants. The constructed coexpression network model showed 1,853 nodes, 5,740 edges, and 142 connected components (correlation coefficient > 0.85). On the basis of the significantly expressed genes in the network, we concluded that the development of salt tolerance is closely related to the activation of $Na^+$ transport by reactive oxygen species signaling and the accumulation of proline in Chinese cabbage.

Constitutive Coexpression of Bacillus Endoxylanase and Trichoderma Endoglucanase Genes in Saccharomyces cerevisiae

  • Lee, Jae-Hyung;Lim, Myung-Ye;Kim, Mi-Jin;Heo, Sun-Yeon;Seo, Jin-Ho;Kim, Yeon-Hee;Nam, Soo-Wan
    • Journal of Microbiology and Biotechnology
    • /
    • v.17 no.12
    • /
    • pp.2076-2080
    • /
    • 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.

Optimized Production of Selenocysteine Containing Formate Dehydrogenase H in Escherichia coli (대장균에서 Selenocysteine을 가지고 있는 Formate Dehydrogenase H의 최적화 생산)

  • Sa, Young-Seung;Kim, Yong-Hwan
    • KSBB Journal
    • /
    • v.26 no.3
    • /
    • pp.189-192
    • /
    • 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.