A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein
A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow...
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| Veröffentlicht in: | Journal of biotechnology 2005, Vol.119 (2), p.133-146 |
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| container_title | Journal of biotechnology |
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| creator | Hedhammar, My Stenvall, Maria Lönneborg, Rosa Nord, Olof Sjölin, Olle Brismar, Hjalmar Uhlén, Mathias Ottosson, Jenny Hober, Sophia |
| description | A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His
6 and Z
basic, and their effect on the expression pattern. |
| doi_str_mv | 10.1016/j.jbiotec.2005.03.024 |
| format | Article |
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6 and Z
basic, and their effect on the expression pattern.</description><identifier>ISSN: 0168-1656</identifier><identifier>ISSN: 1873-4863</identifier><identifier>EISSN: 1873-4863</identifier><identifier>DOI: 10.1016/j.jbiotec.2005.03.024</identifier><identifier>PMID: 15996784</identifier><identifier>CODEN: JBITD4</identifier><language>eng</language><publisher>Lausanne: Elsevier B.V</publisher><subject>Biologi ; Biological and medical sciences ; Biology ; Biotechnology ; Chemical Precipitation ; Cloning, Molecular ; Escherichia coli ; Escherichia coli - cytology ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Flow cytometry ; Flow Cytometry - methods ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; Green Fluorescent Protein (GFP) ; His 6 ; Inclusion bodies ; Inclusion Bodies - chemistry ; Inclusion Bodies - metabolism ; Medicin och hälsovetenskap ; NATURAL SCIENCES ; NATURVETENSKAP ; Protein production levels ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Solubility ; Z basic</subject><ispartof>Journal of biotechnology, 2005, Vol.119 (2), p.133-146</ispartof><rights>2005 Elsevier B.V.</rights><rights>2005 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c517t-e85f788408d0657f686baaa173588af5e9758fe2623d378efd15eb8de6603a1c3</citedby><cites>FETCH-LOGICAL-c517t-e85f788408d0657f686baaa173588af5e9758fe2623d378efd15eb8de6603a1c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jbiotec.2005.03.024$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,314,780,784,885,3550,4024,27923,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=17084573$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15996784$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-8273$$DView record from Swedish Publication Index$$Hfree_for_read</backlink><backlink>$$Uhttp://kipublications.ki.se/Default.aspx?queryparsed=id:111459339$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><creatorcontrib>Hedhammar, My</creatorcontrib><creatorcontrib>Stenvall, Maria</creatorcontrib><creatorcontrib>Lönneborg, Rosa</creatorcontrib><creatorcontrib>Nord, Olof</creatorcontrib><creatorcontrib>Sjölin, Olle</creatorcontrib><creatorcontrib>Brismar, Hjalmar</creatorcontrib><creatorcontrib>Uhlén, Mathias</creatorcontrib><creatorcontrib>Ottosson, Jenny</creatorcontrib><creatorcontrib>Hober, Sophia</creatorcontrib><title>A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein</title><title>Journal of biotechnology</title><addtitle>J Biotechnol</addtitle><description>A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His
6 and Z
basic, and their effect on the expression pattern.</description><subject>Biologi</subject><subject>Biological and medical sciences</subject><subject>Biology</subject><subject>Biotechnology</subject><subject>Chemical Precipitation</subject><subject>Cloning, Molecular</subject><subject>Escherichia coli</subject><subject>Escherichia coli - cytology</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Flow cytometry</subject><subject>Flow Cytometry - methods</subject><subject>Fundamental and applied biological sciences. 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Psychology</topic><topic>Gene Expression</topic><topic>Green Fluorescent Protein (GFP)</topic><topic>His 6</topic><topic>Inclusion bodies</topic><topic>Inclusion Bodies - chemistry</topic><topic>Inclusion Bodies - metabolism</topic><topic>Medicin och hälsovetenskap</topic><topic>NATURAL SCIENCES</topic><topic>NATURVETENSKAP</topic><topic>Protein production levels</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Solubility</topic><topic>Z basic</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hedhammar, My</creatorcontrib><creatorcontrib>Stenvall, Maria</creatorcontrib><creatorcontrib>Lönneborg, Rosa</creatorcontrib><creatorcontrib>Nord, Olof</creatorcontrib><creatorcontrib>Sjölin, Olle</creatorcontrib><creatorcontrib>Brismar, Hjalmar</creatorcontrib><creatorcontrib>Uhlén, Mathias</creatorcontrib><creatorcontrib>Ottosson, Jenny</creatorcontrib><creatorcontrib>Hober, Sophia</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>SwePub</collection><collection>SwePub Articles</collection><collection>SWEPUB Kungliga Tekniska Högskolan</collection><jtitle>Journal of biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hedhammar, My</au><au>Stenvall, Maria</au><au>Lönneborg, Rosa</au><au>Nord, Olof</au><au>Sjölin, Olle</au><au>Brismar, Hjalmar</au><au>Uhlén, Mathias</au><au>Ottosson, Jenny</au><au>Hober, Sophia</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein</atitle><jtitle>Journal of biotechnology</jtitle><addtitle>J Biotechnol</addtitle><date>2005</date><risdate>2005</risdate><volume>119</volume><issue>2</issue><spage>133</spage><epage>146</epage><pages>133-146</pages><issn>0168-1656</issn><issn>1873-4863</issn><eissn>1873-4863</eissn><coden>JBITD4</coden><abstract>A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His
6 and Z
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| ispartof | Journal of biotechnology, 2005, Vol.119 (2), p.133-146 |
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| source | MEDLINE; Elsevier ScienceDirect Journals Complete |
| subjects | Biologi Biological and medical sciences Biology Biotechnology Chemical Precipitation Cloning, Molecular Escherichia coli Escherichia coli - cytology Escherichia coli - genetics Escherichia coli - metabolism Flow cytometry Flow Cytometry - methods Fundamental and applied biological sciences. Psychology Gene Expression Green Fluorescent Protein (GFP) His 6 Inclusion bodies Inclusion Bodies - chemistry Inclusion Bodies - metabolism Medicin och hälsovetenskap NATURAL SCIENCES NATURVETENSKAP Protein production levels Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Solubility Z basic |
| title | A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein |
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