• Title/Summary/Keyword: recombinant E. coli

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Production of Poly(3-hydroxybutyrate) [P(3HB)] with High P(3HB) Content by Recombinant Escherichia coli Harboring the Alcaligenes latus P(3HB) Biosynthesis Genes and the E. coli ftsZ Gene

  • Choi, Jong-Il;Lee, Sang-Yup
    • Journal of Microbiology and Biotechnology
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    • v.9 no.6
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    • pp.722-725
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    • 1999
  • Filamentation-suppressed recombinant Escherichia coli strain harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes and the E. coli ftsZ gene was constructed and cultivated for the production of poly(3-hydroxybutyrate) [P(3HB)] with high concentration and high content. By the pH-stat fed-batch culture of this recombinant E. coli strain XL1-Blue(pJC5), the final cell concentration and P(3HB) concentration obtained in 44.25h were 172.2g cell dry weight/l and 141.9g P(3HB)/l, respectively, resulting in productivity of 3.21g P(3HB)/l-h. More importantly, the P(3HB) content obtained was 82.4 wt %, which was significantly higher than that obtained with the recombinant E. coli harboring only the PHA biosynthesis genes.

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The Structural Characterization of Recombinant Bovine Somatotropin Expressed in Escherichia coli (재조합 소성장호르몬의 구조적 특성)

  • 김정호;김훈주박은숙김준
    • KSBB Journal
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    • v.9 no.2
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    • pp.165-173
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    • 1994
  • In this paper we have described the structural characterization of recombinant bovine somatotropin produced in Escherichia coli. Recombinant bovine somatotropin consists of 191 amino acid residues with a calculated molecular weight of 21,802 Da. For fragmentation of recombinant bovine somatotropin, we have used trypsin, Staphylococcus aureus V8 pretease, CNBr, and mild acid hydrolysis method. Digestion and cleavage with these proteases and chemicals yielded peptides of various size for amino acid sequence determination. The N-terminal sequence analysis was carried out up to thirty residues. Because the design of the recombinant bovine somatotropin gene for expression was such that the coding sequence begins with an initiation codon, AUG, before Ala, the first amino acid of bovine somatotropin, we could expect the initial amino acid as N-formyl Met. But the first amino acid of this protein, expressed in E. coli cells as inclusion bodies, was Ala. And the amino acid composition of RP-HPLC purified recombinant bovine somatotropin was determined and no essencial difference was observed. The amino acid sequence of the recombinant bovine somatotropin was identical to that predicted from its recombinant gene. There was no processing or replacement of amino acid residues in recombinant bovine somatotropin expressed in E. coli. The hydropathy plot of recombinant bovine somatotropin revealed a hydrophobic region at the NH2-terminus and hydrophilic region at the COOH-terminus. The E. coli expression system is thought to be valuable for the expression of recombinant bovine somatotropin because protein was processed to remove the N-terminal Met residue by methionyl-aminopeptidase autonomously.

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Design of Bacterial Vector Systems for the Production of Recombinant Proteins in Escherichia coli

  • Mergulhao;Filipe J.M.;Gabriel A. Monteiro;Joaquim M.S. Cabral;M. Angela Taipa
    • Journal of Microbiology and Biotechnology
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    • v.14 no.1
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    • pp.1-14
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    • 2004
  • More than twenty years have passed since the approval of the first recombinant DNA product for therapeutic use (recombinant human insulin, 1982). However, the biotechnology industry is still facing a shortage of manufacturing capacity due to the increasing demand of therapeutic proteins. This demand has prompted the search for a growing number of biological production systems but, nevertheless, the Gram-negative bacterium Escherichia coli remains one of the most attractive production hosts. This review highlights the most important features and developments of plasmid vector design, emphasizing the different reported strategies for improving the expression and secretion of heterologous proteins using the cellular machinery of E. coli.

Soluble Expression of Human Angiostatin and Endostatin by Maltose Binding Protein (MBP) Fusion in E. coli (Maltose Binding Protein 융합단백질에 의한 인간유래의 앤지오스타틴과 앤도스타틴의 대장균에서 수용성 단백질발현)

  • Paek, Seon-Yeol;Choi, Shin-Geon
    • Journal of Industrial Technology
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    • v.28 no.B
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    • pp.59-63
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    • 2008
  • Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

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Conditions for Stable light Production of Recombinant Escherichia coli Containing Lux Operon and Sensitivity to Toxic Chemicals (Lux operon을 함유한 유전자 재조합 Escherichia coli의 발광 안정화 조건 및 독성물질에 대한 민감성)

  • 배희경;이상민;정윤철;송방호;신평균
    • KSBB Journal
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    • v.17 no.6
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    • pp.571-576
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    • 2002
  • Recombinant E. coli DH5 ${\alpha}$/pSB311 was made by cloning the genes encoding bacterial luciferase and aldehyde substrate proteins from Photohabdus luminescense, to complement defects of Lumistox, which is normally used in bioassays to monitor toxic substances in water environmental systems. The conditions for stable light production by the recombinant strains were investigated with respect to cell growth stage, cell number, and buffer conditions. The optimum growth stage was a middle-exponential stage with an OD$_{660nm}$ value of 0.6-0.7. ADout 10$^{6}$-10$^{7}$ cells per test tube was optimum for stable light emission. The effect of buffer was not significant if an optimum viable cell number was maintained. The bioluminescence of the recombinant E. coli harboring the lux operon of Photohabdus luminescense was not affected by temperature, while the bioluminescence of Lumistox was temperature sensitive. The recombinant E. coli was more sensitive to heavy metals (Cd, Cu, Hg, Zn) than Lumistox, because it does not require high concentrations of NaCl in the buffer.

Comparison of enzymatic activities between the recombinant CHT1 proteins from the hard tick Haemaphysalis longicornis expressed in E. coli and baculovirus-mediated Sf 9 cells (E. coli와 baculovirus-mediated Sf 9 세포에서 발현된 진드기 H. longicornis의 CHT1 단백의 효소활성 비교)

  • You, Myung-Jo;Fujisaki, Kozo
    • Korean Journal of Veterinary Research
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    • v.43 no.1
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    • pp.139-144
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    • 2003
  • A chitinase cDNA named CHT1 was cloned from the hard tick, Haemaphysalis longicornis, and the enzymatic properties of its recombinant proteins were characterized. The CHT1 cDNA encodes 930 amino-acid (aa) residues including a 22 aa putative signal peptide, with the calculated molecular mass of the putative mature protein 104 kDa. The E coli-expressed rCHT1 exhibited weak chitinolytic activity against $4MU-(GlcNAc)_3$. The rCHT1 protein with higher activity was obtained using recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), which expresses rCHT1 under polyhedrin promoter. These findings suggest that the rCHT1 expressed in baculovirus-mediated Sf 9 cells has a high activity than E coli-expressed rCHT1.

Stability of Recombinant Plasmids Carrying the stb Locus of E. coli IncFII NR1 Plasmid in E. coli and Yeast (대장균과 효모에서 Escherichia coli IncFII NR1 플라스미드의 stb 좌위를 포함하는 재조합 플라스미드의 안정성에 관한 연구)

  • Chung, Kung-Sook;Kim, Choon-Kwang;Kim, Kyu-Won
    • Korean Journal of Microbiology
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    • v.31 no.1
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    • pp.37-43
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    • 1993
  • The effect of stb locus of E. COLI IncFII plasmid NR1 on the stability of chimeric plasmids was investigated. First, we have isolated the stability locus (stb) from E. coli NR1 plasmid and then inserted into the three different vectors, pUC8, YRp17 and YEp24. By examining their stability in E. coli and yeast, we showed that the recombinant plasmids containing stb locus were resonably stable. Also, by comparing the amounts of the rDNA fragments per haploid genome with those of the plasmid fragments, we showed they copy number of recombinant plasmids was not increased. Consequently, the stb locus of E. coli IncFII plasmid NR1 stabilized the chimeric plasmids but did not affect the replication or copy number of plasmids.

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Molecular Cloning of Pseudomonas sp.Inulinase Gene and its Expresstion in E. coli (Pseudomonas sp. Inulinase 유전자의 클로닝 및 Escherichia coli에서의 발현)

  • 엄수정;권영만;최용진
    • Microbiology and Biotechnology Letters
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    • v.23 no.5
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    • pp.550-555
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    • 1995
  • A strain of Pseudomonas sp. isolated from soil was shown to produce a high level of extracellular endo-inulinase. In this work, the endo-inulinase gene (inu1) of the bacterial strain was cloned into the plasmid pBR322 by using EcoRI restriction endonuclease and E. coli HB101 as a host strain. One out of 7, 000 transformants obtained from the above cloning experiment formed a clear zone around its colony on the selective medium supplemented with 2.0% inulin after a prolonged incubation at 37$\circ$C and subsequent cold shock treatment. The functional clone was found to carry a recombinant plasmid (pKMG50) with a 3.7 kb genomic insert containing the genetic information for the inulinase activity. The inulinase from E. coli HB101/pKMG50 was proved to be an endo-acting enzyme and produced constitutively in the recombinant E. coli cells. Zymogram of the enzyme from the recombinant cells with inulin substrate indicated that the molecular mass of the active protein was 190 Kd, while that of the endo-inulinase from the Pseudomonas strain was 170 Kd. This size discrepancy suggested that the inulinase from the recombinant E. coli HB101 cells might be the initial product of translation, not the mature form produced in the strain of Pseudomonas sp..

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Proteome Analysis of Overproduction of Recombinant Protein in Escherichia coli by Fed-Batch Fermentations

  • Han, Mi-Jeong;Choe, Jong-Hyeon;Jeong, Gi-Jun;Yu, Jong-Sin;Lee, Sang-Yeop
    • 한국생물공학회:학술대회논문집
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    • 2001.11a
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    • pp.750-753
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    • 2001
  • Proteomics is a formalized approach for obtaining a rapid snap-shot of the protein complement of a tissue, cell or cell component. Such an approach is powerful in that it allows a parallel assessment of temporal protein fluxes. This is an important concept in view of the dynamic nature of protein expression. Undoubtedly, changes in protein expression are essential in any study aimed at investigating cellular networks. In this study, we analyzed and compared the proteomes of recombinant E. coli strain before and after induction. Proteome expression patterns of recombinant E. coli were resolved on 2D-gels, and the variations in the relative expression level of particular proteins were examined using software-aided protein quantification tool. We observed above 800 spots on a 2D-gel using Melanie II software. Many proteins which involved in chaperones were significantly up-regulated in recombinant E. coli. Therefore, it could be concluded that the expression of recombinant protein in E. coli acted as a stress to the cells, which change cells ability to synthesize proteins and induced the expression of various protective proteins.

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Characterization of the Pediocin Operon of Pediococcus acidilactici K10 and Expression of His-Tagged Recombinant Pediocin PA-1 in Escherichia coli

  • MOON GI SEONG;PYUN YU RYANG;KIM WANG JUNE
    • Journal of Microbiology and Biotechnology
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    • v.15 no.2
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    • pp.403-411
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    • 2005
  • The relationship between plasmid (~9.5 kb) and pediocin PA-1 in P. acidilactici K10 was confirmed by plasmid curing. The pediocin operon of P. acidilactici K10 was amplified by PCR (polymerase chain reaction), and the nucleotide sequence was analyzed. The sequence of the pediocin operon of P. acidilactici K10 was similar to those of P. acidilactici strains producing pediocin PA-1/ AcH. For the expression of pediocin PA-1 in E. coli, a pQEPED (pQE-30 Xa::mature pedA) was constructed. His-tagged recombinant pediocin PA-1 (-6.5 kDa) was translated by cell-free in vitro transcription and translation using pQEPED as a DNA template. Theresult of slot blotting assay showed that transcription of recombinant pedA in E. coli M15 was induced by the addition of isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) at the final concentration of 1 mM. Although the recombinant pediocin PA-1 inhibited the growth of E. coli, it was expressed in the host strain and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography under denaturing condition. This is the first report for the production and one-step purification of biologically active recombinant pediocin PA-1 in E. coli.