1,720,994 research outputs found
Proteome profiling and its use in metabolic and, cellular engineering
No full textProteome profiling of microorganisms makes it possible to generate valuable knowledge that can be used for the development of metabolic and cellular engineering strategies, which consequently are used to enhance the yield and productivity of native or foreign bioproducts and to modify cellular properties to improve mid-stream and down-stream processes. Advances in two-dimensional gel electrophoresis technology combined with mass spectrometry allow the creation of global scale proteome contents which can be used to elucidate valuable information on the dynamics of the metabolic, signaling and regulatory networks apart from understanding the physiological changes. In this paper, we review the approaches of exploiting the proteome profiling results to the development of the strategies for the metabolic and cellular engineering of microorganisms.This work was supported by the National Research Laboratory
Program (2000-N-NL-01-C-237) of the Ministry of
Science and Technology (MOST), the Basic Industrial Research
Project from the Ministry of Commerce, Industry
and Energy, Center for Ultramicrochemical Process Systems,
and by the Brain Korea 21 project
Microbial small heat shock proteins and their use in biotechnology
No full textSmall heat shock proteins (sHsps) exist in almost all organisms. Most organisms have more than one sHsp, but their number can be as high as 65, as found in the eukaryote, Vitis vinifera. The function of sHsps is well-known; they confer thermotolerance to cellular cultures and proteins in cellular extracts during prolonged incubations at elevated temperatures. This demonstrates the ability of sHsps to protect cellular proteins, and to maintain cellular viability under conditions of intensive stress, such as heat shock or chemical treatments. sHsps have several properties that distinguish them from heat shock proteins (Hsps): they function as ATP-independent chaperones, require the flexible assembly and reassembly of oligomeric complex structures for their activation, and exhibit a wide range of substrate-binding capacities. Recent studies indicate that sHsps have important biological functions in thermostability, disaggregation, and proteolysis inhibition. These functions can be harnessed for various applications, including nanobiotechnology, proteomics, bioproduction, and bioseparation. In this review, we discuss the properties and diversity of microbial sHsps, as well as their potential uses in the biotechnology industry. (c) 2008 Elsevier Inc. All rights reservedThis work was supported by the Korean Systems Biology Research
Grant (M10309020000-03B5002-00000) of the Ministry of Education,
Science and Technology through the KOSEF. Further supports by LG
Chem Chair Professorship, Microsoft, and by the KOSEF through the
Center for Ultramicrochemical Process Systems are appreciated
Proteome: Past, Present, and Future Prospects
Full text linkProteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects.This work was supported by a Korean Systems Biology Research
Grant (M10309020000-03B5002-00000) of the Ministry of Science and
Technology. Further support by the LG Chem Chair Professorship, the
IBM SUR program, Microsoft, KOSEF through the Center for Ultramicrochemical
Process Systems, and the Brain Korea 21 project is
appreciated
Proteome analysis of metabolically engineered Escherichia coli producing poly(3-hydroxybutyrate)
No full textRecombinant Escherichia call strains harboring heterologous polyhydroxyalkanoate (PHA) biosynthesis genes were shown to accumulate unusually large amounts of PHA, In the present study, integrated cellular responses of metabolically engineered E. coli to the accumulation of poly(3-hydroxybutyrate) (PHB) in the early stationary phase were analyzed at the protein level by two-dimensional gel electrophoresis, Out of 20 proteins showing altered expression levels with the accumulation of PHB, 13 proteins were identified with the aid of mass spectrometry, Three heat shock proteins, GroEL, GroES, and DnaK, were significantly up-regulated in PHB-accumulating cells, Proteins which play essential roles in protein biosynthesis were unfavorably influenced by the accumulation of PHB. Cellular demand for the large amount of acetyl coenzyme A and NADPH for the PHB biosynthesis resulted in the increased synthesis of two enzymes of the glycolytic pathway and one enzyme of the Entner-Doudoroff pathway. The expression of the yfiD gene encoding a 14,3-kDa protein, which is known to be produced at low pH, was greatly induced with the accumulation of PHB. Therefore, it could be concluded that the accumulation of PHB in E. coli acted as a stress on the cells, which reduced the cells' ability to synthesize proteins and induced the expression of various protective proteins.We thank Jong Shin Yu at the Korea Basic Science Institute for his
generous help in the MALDI-TOF analysis.
This work was supported by the National Research Laboratory program
of the Korean Ministry of Science and Technology and by the
First Young Scientist’s Award to S. Y. Lee by the President of Korea
Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation
No full textEscherichia coli BL21 (DE3) and W3110 strains, belonging to the family B and K-12, respectively, have been most widely employed for recombinant protein production. During the excretory production of recombinant proteins by high cell density cultivation (HCDC) of these strains, other native E. coli proteins were also released. Thus, we analyzed the extracellular proteomes of E. coli BL21 (DE3) and W3110 during HCDC. E. coli BL21 (DE3) released more than twice the amount of protein compared with W3110 during HCDC. A total of 204 protein spots including 83 nonredundant proteins were unambiguously identified by 2-DE and MS. Of these, 32 proteins were conserved in the two strains, while 20 and 33 strain-specific proteins were identified for E. coli BL21 (DE3) and W3110, respectively. More than 70% of identified proteins were found to be of periplasmic origin. The outer membrane proteins, OmpA and OmpF, were most abundant. Two strains showed much different patterns in their released proteins. Also, cell density-dependent variations in the released proteins were observed in both strains. These findings summarized as reference proteome maps will be useful for studying protein release in further detail, and provide new strategies for enhanced excretory production of recombinant proteins.This work was supported by the Korean Systems Biology Research
Grant (M10309020000-03B5002-00000) of the Ministry
of Science and Technology. Further supports by LG Chem Chair
Professorship, IBM SUR Program, Microsoft, and by the KOSEF
through the Center for Ultramicrochemical Process Systems are
appreciated
Proteome-level responses of Escherichia coli to long-chain fatty acids and use of fatty acid inducible promoter in protein production
No full textIn Escherichia coli, a long-chain acyl-CoA is a regulatory signal that modulates gene expression through its binding to a transcription factor FadR. In this study, comparative proteomic analysis of E. coli in the presence of glucose and oleic acid was performed to understand cell physiology in response to oleic acid. Among total of 52 proteins showing altered expression levels with oleic acid presence, 9 proteins including AldA, Cdd, FadA, FadB, FadL, MalE, RbsB, Udp, and YccU were newly synthesized. Among the genes that were induced by oleic acid, the promoter of the aldA gene was used for the production of a green fluorescent protein (GFP). Analysis of fluorescence intensities and confocal microscopic images revealed that soluble GFP was highly expressed under the control of the aldA promoter. These results suggest that proteomics is playing an important role not only in biological research but also in various biotechnological applications. Copyright (c) 2008.We thank Z. W. Lee (Korea Basic Science Institute, Daejeon,
Republic of Korea) for his help with the confocal microscopy.
This work was supported by the Korean Systems Biology Research
Grant (M10309020000-03B5002-00000) of the Ministry
of Science and Technology. Further supports by LG
Chem Chair Professorship, IBM SUR program, Microsoft,
and by the KOSEF through the Center for Ultramicrochemical
Process Systems are appreciated
Combined transcriptome and proteome analysis of Escherichia coli during high cell density culture
No full textCombined transcriptome and proteome analysis was carried out to understand metabolic and physiological changes of Escherichia coli during the high cell density cultivation (HCDC). The expression of genes of TCA cycle enzymes, NADH dehydrogenase and ATPase, was up-regulated during the exponential fed-batch period and was down-regulated afterward. However, expression of most of the genes involved in glycolysis and pentose phosphate pathway was up-regulated at the stationary phase. The expression of most of amino acid biosynthesis genes was down-regulated as cell density increased, which seems to be the major reason for the reduced specific productivity of recombinant proteins during HCDC. The expression of chaperone genes increased with cell density, suggesting that the high cell density condition itself can be stressful to the cells. Severe competition for oxygen at high cell density seemed to make cells use cytochrome bd, which is less efficient but has a high oxygen affinity than cytochrome boa. Population cell density itself strongly affected the expression of porin protein genes, especially ompF, and hence the permeability of the outer membrane. Expression of phosphate starvation genes was most strongly up-regulated toward the end of cultivation. It was also found that sigma(E) (rpoE) plays a more important role than us (rpoS) at the stationary phase of HCDC. These findings should be invaluable in designing metabolic engineering and fermentation strategies for the production of recombinant proteins and metabolites by HCDC of E. coli. (C) 2003 Wiley Periodicals, Inc
Roles and applications of small heat shock proteins in the production of recombinant proteins in Escherichia coli
No full textProteome profiling of the inclusion body (IB) fraction of recombinant proteins produced in Escherichia coli suggested that two small heat shock proteins, IbpA and lbpB, are the major proteins associated with IBs. In this study, we demonstrate that IbpA and IbpB facilitate the production of recombinant proteins in E. coli and play important roles in protecting recombinant proteins from degradation by cytoplasmic proteases. We examined the cytosolic production, and Tat- or Sec-dependent secretion of the enhanced green fluorescent protein (EGFP) in wild type, ibpAB(-) mutant, and ibpAB-amplified E. coli strains. Analysis of fluorescence histograms and confocal microscopic imaging revealed that over-expression of the ibpA and/or ibpB genes enhanced cytosolic EGFP production whereas knocking out the ibpAB genes enhanced secretory production. This strategy seems to be generally applicable as it was successfully employed for the enhanced cytosolic or secretory production of several other recombinant proteins in E. coli. (C) 2004 Wiley Periodicals, Inc.We thank E. Laskowska (Gdan´sk, Poland) for anti-IbpA/B sera and
also Z. W. Lee (Korea Basic Science Institute, Daejeon, Republic of
Korea) for his help with the confocal microscopy. Further support by
LG Chem Chair Professorship and BK21 program is appreciated
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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