Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.
Identifieur interne : 001446 ( Main/Exploration ); précédent : 001445; suivant : 001447Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.
Auteurs : Mark Ellis [Royaume-Uni] ; Pareshkumar Patel [Royaume-Uni] ; Marjory Edon [France] ; Walter Ramage [Royaume-Uni] ; Robert Dickinson [Australie] ; David P. Humphreys [Royaume-Uni]Source :
- Biotechnology progress [ 1520-6033 ] ; 2017.
Descripteurs français
- KwdFr :
- Anticorps monoclonaux humanisés (biosynthèse), Anticorps monoclonaux humanisés (composition chimique), Anticorps monoclonaux humanisés (isolement et purification), Escherichia coli (composition chimique), Escherichia coli (génétique), Fermentation (MeSH), Fragments Fab d'immunoglobuline (biosynthèse), Fragments Fab d'immunoglobuline (composition chimique), Fragments Fab d'immunoglobuline (isolement et purification), Humains (MeSH), Ingénierie des protéines (MeSH), Peptide hydrolases (composition chimique), Peptide hydrolases (isolement et purification), Pliage des protéines (MeSH), Périplasme (composition chimique), Périplasme (génétique).
- MESH :
- biosynthèse : Anticorps monoclonaux humanisés, Fragments Fab d'immunoglobuline.
- composition chimique : Anticorps monoclonaux humanisés, Escherichia coli, Fragments Fab d'immunoglobuline, Peptide hydrolases, Périplasme.
- génétique : Escherichia coli, Périplasme.
- isolement et purification : Anticorps monoclonaux humanisés, Fragments Fab d'immunoglobuline, Peptide hydrolases.
- Fermentation, Humains, Ingénierie des protéines, Pliage des protéines.
English descriptors
- KwdEn :
- Antibodies, Monoclonal, Humanized (biosynthesis), Antibodies, Monoclonal, Humanized (chemistry), Antibodies, Monoclonal, Humanized (isolation & purification), Escherichia coli (chemistry), Escherichia coli (genetics), Fermentation (MeSH), Humans (MeSH), Immunoglobulin Fab Fragments (biosynthesis), Immunoglobulin Fab Fragments (chemistry), Immunoglobulin Fab Fragments (isolation & purification), Peptide Hydrolases (chemistry), Peptide Hydrolases (isolation & purification), Periplasm (chemistry), Periplasm (genetics), Protein Engineering (MeSH), Protein Folding (MeSH).
- MESH :
- chemical , biosynthesis : Antibodies, Monoclonal, Humanized, Immunoglobulin Fab Fragments.
- chemical , chemistry : Antibodies, Monoclonal, Humanized, Immunoglobulin Fab Fragments, Peptide Hydrolases.
- chemical , isolation & purification : Antibodies, Monoclonal, Humanized, Immunoglobulin Fab Fragments, Peptide Hydrolases.
- chemistry : Escherichia coli, Periplasm.
- genetics : Escherichia coli, Periplasm.
- Fermentation, Humans, Protein Engineering, Protein Folding.
Abstract
Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.
DOI: 10.1002/btpr.2393
PubMed: 27790865
Affiliations:
Links toward previous steps (curation, corpus...)
Le document en format XML
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Humphreys</name><affiliation wicri:level="1"><nlm:affiliation>Discovery Research, Protein Sciences, UCB Pharma, 208 Bath Road, Slough, Berkshire, SL1 3WE, U.K.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Discovery Research, Protein Sciences, UCB Pharma, 208 Bath Road, Slough, Berkshire, SL1 3WE</wicri:regionArea><wicri:noRegion>SL1 3WE</wicri:noRegion></affiliation></author></analytic><series><title level="j">Biotechnology progress</title><idno type="eISSN">1520-6033</idno><imprint><date when="2017" type="published">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Antibodies, Monoclonal, Humanized (biosynthesis)</term><term>Antibodies, Monoclonal, Humanized (chemistry)</term><term>Antibodies, Monoclonal, Humanized (isolation & purification)</term><term>Escherichia coli (chemistry)</term><term>Escherichia coli (genetics)</term><term>Fermentation (MeSH)</term><term>Humans (MeSH)</term><term>Immunoglobulin Fab Fragments (biosynthesis)</term><term>Immunoglobulin Fab Fragments (chemistry)</term><term>Immunoglobulin Fab Fragments (isolation & purification)</term><term>Peptide Hydrolases (chemistry)</term><term>Peptide Hydrolases (isolation & purification)</term><term>Periplasm (chemistry)</term><term>Periplasm (genetics)</term><term>Protein Engineering (MeSH)</term><term>Protein Folding (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Anticorps monoclonaux humanisés (biosynthèse)</term><term>Anticorps monoclonaux humanisés (composition chimique)</term><term>Anticorps monoclonaux humanisés (isolement et purification)</term><term>Escherichia coli (composition chimique)</term><term>Escherichia coli (génétique)</term><term>Fermentation (MeSH)</term><term>Fragments Fab d'immunoglobuline (biosynthèse)</term><term>Fragments Fab d'immunoglobuline (composition chimique)</term><term>Fragments Fab d'immunoglobuline (isolement et purification)</term><term>Humains (MeSH)</term><term>Ingénierie des protéines (MeSH)</term><term>Peptide hydrolases (composition chimique)</term><term>Peptide hydrolases (isolement et purification)</term><term>Pliage des protéines (MeSH)</term><term>Périplasme (composition chimique)</term><term>Périplasme (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Antibodies, Monoclonal, Humanized</term><term>Immunoglobulin Fab Fragments</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Antibodies, Monoclonal, Humanized</term><term>Immunoglobulin Fab Fragments</term><term>Peptide Hydrolases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Antibodies, Monoclonal, Humanized</term><term>Immunoglobulin Fab Fragments</term><term>Peptide Hydrolases</term></keywords><keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Anticorps monoclonaux humanisés</term><term>Fragments Fab d'immunoglobuline</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Escherichia coli</term><term>Periplasm</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Anticorps monoclonaux humanisés</term><term>Escherichia coli</term><term>Fragments Fab d'immunoglobuline</term><term>Peptide hydrolases</term><term>Périplasme</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term><term>Periplasm</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Escherichia coli</term><term>Périplasme</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Anticorps monoclonaux humanisés</term><term>Fragments Fab d'immunoglobuline</term><term>Peptide hydrolases</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Fermentation</term><term>Humans</term><term>Protein Engineering</term><term>Protein Folding</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Fermentation</term><term>Humains</term><term>Ingénierie des protéines</term><term>Pliage des protéines</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD<sub>600</sub> 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">27790865</PMID><DateCompleted><Year>2017</Year><Month>12</Month><Day>12</Day></DateCompleted><DateRevised><Year>2018</Year><Month>02</Month><Day>04</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1520-6033</ISSN><JournalIssue CitedMedium="Internet"><Volume>33</Volume><Issue>1</Issue><PubDate><Year>2017</Year><Month>01</Month></PubDate></JournalIssue><Title>Biotechnology progress</Title><ISOAbbreviation>Biotechnol Prog</ISOAbbreviation></Journal><ArticleTitle>Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.</ArticleTitle><Pagination><MedlinePgn>212-220</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1002/btpr.2393</ELocationID><Abstract><AbstractText>Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD<sub>600</sub> 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.</AbstractText><CopyrightInformation>© 2016 American Institute of Chemical Engineers.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Ellis</LastName><ForeName>Mark</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Discovery Research, Protein Sciences, UCB Pharma, 208 Bath Road, Slough, Berkshire, SL1 3WE, U.K.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Patel</LastName><ForeName>Pareshkumar</ForeName><Initials>P</Initials><AffiliationInfo><Affiliation>Lonza Biologics plc, 228 Bath Road GB-Slough, Berkshire, SL1 4DX, U.K.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Edon</LastName><ForeName>Marjory</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Novasep, 5 chemin du Pilon, St Maurice de Beynost, Miribel, 01708, France.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Ramage</LastName><ForeName>Walter</ForeName><Initials>W</Initials><AffiliationInfo><Affiliation>NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, U.K.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Dickinson</LastName><ForeName>Robert</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>CSL Limited, 45 Poplar Road, Parkville, Vic, 3052, Australia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Humphreys</LastName><ForeName>David P</ForeName><Initials>DP</Initials><AffiliationInfo><Affiliation>Discovery Research, Protein Sciences, UCB Pharma, 208 Bath Road, Slough, Berkshire, SL1 3WE, U.K.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2016</Year><Month>11</Month><Day>17</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biotechnol Prog</MedlineTA><NlmUniqueID>8506292</NlmUniqueID><ISSNLinking>1520-6033</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D061067">Antibodies, Monoclonal, Humanized</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D007140">Immunoglobulin Fab Fragments</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.4.-</RegistryNumber><NameOfSubstance UI="D010447">Peptide Hydrolases</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D061067" MajorTopicYN="N">Antibodies, Monoclonal, Humanized</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004926" MajorTopicYN="N">Escherichia coli</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005285" MajorTopicYN="N">Fermentation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D006801" MajorTopicYN="N">Humans</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D007140" MajorTopicYN="N">Immunoglobulin Fab Fragments</DescriptorName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010447" MajorTopicYN="N">Peptide Hydrolases</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D019897" MajorTopicYN="N">Periplasm</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015202" MajorTopicYN="N">Protein Engineering</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017510" MajorTopicYN="N">Protein Folding</DescriptorName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="Y">DsbC</Keyword><Keyword MajorTopicYN="Y">Escherichia coli</Keyword><Keyword MajorTopicYN="Y">Fab</Keyword><Keyword MajorTopicYN="Y">Tsp</Keyword><Keyword MajorTopicYN="Y">fermentation</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2015</Year><Month>11</Month><Day>10</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2016</Year><Month>10</Month><Day>19</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2016</Year><Month>10</Month><Day>30</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2017</Year><Month>12</Month><Day>13</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2016</Year><Month>10</Month><Day>30</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">27790865</ArticleId><ArticleId IdType="doi">10.1002/btpr.2393</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Australie</li><li>France</li><li>Royaume-Uni</li></country><region><li>Auvergne-Rhône-Alpes</li><li>Rhône-Alpes</li></region></list><tree><country name="Royaume-Uni"><noRegion><name sortKey="Ellis, Mark" sort="Ellis, Mark" uniqKey="Ellis M" first="Mark" last="Ellis">Mark Ellis</name></noRegion><name sortKey="Humphreys, David P" sort="Humphreys, David P" uniqKey="Humphreys D" first="David P" last="Humphreys">David P. Humphreys</name><name sortKey="Patel, Pareshkumar" sort="Patel, Pareshkumar" uniqKey="Patel P" first="Pareshkumar" last="Patel">Pareshkumar Patel</name><name sortKey="Ramage, Walter" sort="Ramage, Walter" uniqKey="Ramage W" first="Walter" last="Ramage">Walter Ramage</name></country><country name="France"><region name="Auvergne-Rhône-Alpes"><name sortKey="Edon, Marjory" sort="Edon, Marjory" uniqKey="Edon M" first="Marjory" last="Edon">Marjory Edon</name></region></country><country name="Australie"><noRegion><name sortKey="Dickinson, Robert" sort="Dickinson, Robert" uniqKey="Dickinson R" first="Robert" last="Dickinson">Robert Dickinson</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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| HfdSelect -Kh $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd \
| NlmPubMed2Wicri -a PoplarV1
| This area was generated with Dilib version V0.6.37. |  |