Amino acid content of recombinant proteins influences the metabolic burden response
Recombinant protein production in Escherichia coli often results in a dramatic cellular stress response best characterized by a decrease in overall cell fitness. We determined that the primary sequence (the amino acid sequence) of the recombinant protein alone plays an important role in mitigating t...
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| Veröffentlicht in: | Biotechnology and bioengineering 2005-04, Vol.90 (1), p.116-126 |
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| creator | Bonomo, Jeanne Gill, Ryan T. |
| description | Recombinant protein production in Escherichia coli often results in a dramatic cellular stress response best characterized by a decrease in overall cell fitness. We determined that the primary sequence (the amino acid sequence) of the recombinant protein alone plays an important role in mitigating this response. To do so, we created two polypeptides, modeled after the 39‐40 amino acid Defensin class of proteins, which contained exclusively the five least (PepAA; His, Trp, Tyr, Phe, Met), or most (PepCO: Ala, Glu, Gln, Asp, Asn) abundant amino acids in E. coli. We determined that overexpression of PepAA resulted in a drastic decrease in growth rate compared to overexpression of PepCO, our model Defensin protein MGD‐1, or the 26 amino acid polypeptide contained within the pET‐3d vector backbone. We further determined, using Affymetrix E. coli gene chips, that differences among the whole‐genome transcriptional responses of these model systems were best characterized by altered expression of genes whose products are involved in translation, transport, or metabolic functions as opposed to stress response genes. Based on these results, we confirmed that translation efficiency was significantly reduced in cells overexpressing PepAA compared with the other model polypeptides evaluated. © 2005 Wiley Periodicals, Inc. |
| doi_str_mv | 10.1002/bit.20436 |
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We determined that the primary sequence (the amino acid sequence) of the recombinant protein alone plays an important role in mitigating this response. To do so, we created two polypeptides, modeled after the 39‐40 amino acid Defensin class of proteins, which contained exclusively the five least (PepAA; His, Trp, Tyr, Phe, Met), or most (PepCO: Ala, Glu, Gln, Asp, Asn) abundant amino acids in E. coli. We determined that overexpression of PepAA resulted in a drastic decrease in growth rate compared to overexpression of PepCO, our model Defensin protein MGD‐1, or the 26 amino acid polypeptide contained within the pET‐3d vector backbone. We further determined, using Affymetrix E. coli gene chips, that differences among the whole‐genome transcriptional responses of these model systems were best characterized by altered expression of genes whose products are involved in translation, transport, or metabolic functions as opposed to stress response genes. Based on these results, we confirmed that translation efficiency was significantly reduced in cells overexpressing PepAA compared with the other model polypeptides evaluated. © 2005 Wiley Periodicals, Inc.</description><identifier>ISSN: 0006-3592</identifier><identifier>EISSN: 1097-0290</identifier><identifier>DOI: 10.1002/bit.20436</identifier><identifier>PMID: 15736162</identifier><identifier>CODEN: BIBIAU</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>Amino Acid Sequence ; Amino acids ; Amino Acids - biosynthesis ; Amino Acids - chemistry ; Amino Acids - genetics ; Bacteria ; Biological and medical sciences ; Biotechnology ; Defensins - biosynthesis ; Defensins - chemistry ; Defensins - genetics ; Escherichia coli ; Escherichia coli - chemistry ; Escherichia coli - physiology ; Escherichia coli Proteins - biosynthesis ; Escherichia coli Proteins - chemistry ; Escherichia coli Proteins - genetics ; Fundamental and applied biological sciences. Psychology ; Gene expression ; Gene Expression Regulation, Bacterial - physiology ; Genetic engineering ; Genetic technics ; metabolic burden ; Metabolism ; Methods. Procedures. 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Bioeng</addtitle><description>Recombinant protein production in Escherichia coli often results in a dramatic cellular stress response best characterized by a decrease in overall cell fitness. We determined that the primary sequence (the amino acid sequence) of the recombinant protein alone plays an important role in mitigating this response. To do so, we created two polypeptides, modeled after the 39‐40 amino acid Defensin class of proteins, which contained exclusively the five least (PepAA; His, Trp, Tyr, Phe, Met), or most (PepCO: Ala, Glu, Gln, Asp, Asn) abundant amino acids in E. coli. We determined that overexpression of PepAA resulted in a drastic decrease in growth rate compared to overexpression of PepCO, our model Defensin protein MGD‐1, or the 26 amino acid polypeptide contained within the pET‐3d vector backbone. We further determined, using Affymetrix E. coli gene chips, that differences among the whole‐genome transcriptional responses of these model systems were best characterized by altered expression of genes whose products are involved in translation, transport, or metabolic functions as opposed to stress response genes. Based on these results, we confirmed that translation efficiency was significantly reduced in cells overexpressing PepAA compared with the other model polypeptides evaluated. © 2005 Wiley Periodicals, Inc.</description><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Amino Acids - biosynthesis</subject><subject>Amino Acids - chemistry</subject><subject>Amino Acids - genetics</subject><subject>Bacteria</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Defensins - biosynthesis</subject><subject>Defensins - chemistry</subject><subject>Defensins - genetics</subject><subject>Escherichia coli</subject><subject>Escherichia coli - chemistry</subject><subject>Escherichia coli - physiology</subject><subject>Escherichia coli Proteins - biosynthesis</subject><subject>Escherichia coli Proteins - chemistry</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene expression</subject><subject>Gene Expression Regulation, Bacterial - physiology</subject><subject>Genetic engineering</subject><subject>Genetic technics</subject><subject>metabolic burden</subject><subject>Metabolism</subject><subject>Methods. Procedures. Technologies</subject><subject>Modification of gene expression level</subject><subject>Molecular Sequence Data</subject><subject>Oxidative Stress - physiology</subject><subject>Protein Engineering - methods</subject><subject>Proteins</subject><subject>recombinant protein production</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Structure-Activity Relationship</subject><subject>transcriptional profiling</subject><subject>translation efficiency</subject><issn>0006-3592</issn><issn>1097-0290</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkV9rFDEUxYModq0--AVkEBR8mDY3mSSTx1q0LbQqdMW-hUz2DqbOJNtkBu23N9tdLRTEp8uF3zn3zyHkJdADoJQddn46YLTh8hFZANWqpkzTx2RBKZU1F5rtkWc5X5dWtVI-JXsgFJcg2YJcHo0-xMo6v6pcDBOGqYp9ldDFsfPBlnad4oQ-5MqHfpgxOMzV9B2rESfbxcG7qpvTCkMR5XUMGZ-TJ70dMr7Y1X3y9eOH5fFpff755Oz46Lx2QjBZa2i0Q2CibblmsqEAHIUVXddyRWnLGr1StgPdUw68A6lkwxXyRlPAXlq-T95ufcuGNzPmyYw-OxwGGzDO2UglWNsI_l8QtFJAhSjg6wfgdZxTKEcYBlxJJmDj9m4LuRRzTtibdfKjTbcGqNnkYUoe5i6Pwr7aGc7diKt7chdAAd7sAJudHfpkg_P5npOS01ZvjA633E8_4O2_J5r3Z8s_o-utwucJf_1V2PSjPIYrYb59OjH6YgmXX664ueK_ATZ_rhY</recordid><startdate>20050405</startdate><enddate>20050405</enddate><creator>Bonomo, Jeanne</creator><creator>Gill, Ryan T.</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><general>Wiley</general><general>Wiley Subscription Services, Inc</general><scope>BSCLL</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7U5</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7QL</scope><scope>7X8</scope></search><sort><creationdate>20050405</creationdate><title>Amino acid content of recombinant proteins influences the metabolic burden response</title><author>Bonomo, Jeanne ; Gill, Ryan T.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5526-9149ce12588392640113e5a5bb837008249d7ab19f0313b1676437e34901ef6a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Amino Acid Sequence</topic><topic>Amino acids</topic><topic>Amino Acids - biosynthesis</topic><topic>Amino Acids - chemistry</topic><topic>Amino Acids - genetics</topic><topic>Bacteria</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Defensins - biosynthesis</topic><topic>Defensins - chemistry</topic><topic>Defensins - genetics</topic><topic>Escherichia coli</topic><topic>Escherichia coli - chemistry</topic><topic>Escherichia coli - physiology</topic><topic>Escherichia coli Proteins - biosynthesis</topic><topic>Escherichia coli Proteins - chemistry</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene expression</topic><topic>Gene Expression Regulation, Bacterial - physiology</topic><topic>Genetic engineering</topic><topic>Genetic technics</topic><topic>metabolic burden</topic><topic>Metabolism</topic><topic>Methods. Procedures. Technologies</topic><topic>Modification of gene expression level</topic><topic>Molecular Sequence Data</topic><topic>Oxidative Stress - physiology</topic><topic>Protein Engineering - methods</topic><topic>Proteins</topic><topic>recombinant protein production</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Structure-Activity Relationship</topic><topic>transcriptional profiling</topic><topic>translation efficiency</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bonomo, Jeanne</creatorcontrib><creatorcontrib>Gill, Ryan T.</creatorcontrib><collection>Istex</collection><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>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>MEDLINE - Academic</collection><jtitle>Biotechnology and bioengineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bonomo, Jeanne</au><au>Gill, Ryan T.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Amino acid content of recombinant proteins influences the metabolic burden response</atitle><jtitle>Biotechnology and bioengineering</jtitle><addtitle>Biotechnol. Bioeng</addtitle><date>2005-04-05</date><risdate>2005</risdate><volume>90</volume><issue>1</issue><spage>116</spage><epage>126</epage><pages>116-126</pages><issn>0006-3592</issn><eissn>1097-0290</eissn><coden>BIBIAU</coden><abstract>Recombinant protein production in Escherichia coli often results in a dramatic cellular stress response best characterized by a decrease in overall cell fitness. We determined that the primary sequence (the amino acid sequence) of the recombinant protein alone plays an important role in mitigating this response. To do so, we created two polypeptides, modeled after the 39‐40 amino acid Defensin class of proteins, which contained exclusively the five least (PepAA; His, Trp, Tyr, Phe, Met), or most (PepCO: Ala, Glu, Gln, Asp, Asn) abundant amino acids in E. coli. We determined that overexpression of PepAA resulted in a drastic decrease in growth rate compared to overexpression of PepCO, our model Defensin protein MGD‐1, or the 26 amino acid polypeptide contained within the pET‐3d vector backbone. We further determined, using Affymetrix E. coli gene chips, that differences among the whole‐genome transcriptional responses of these model systems were best characterized by altered expression of genes whose products are involved in translation, transport, or metabolic functions as opposed to stress response genes. Based on these results, we confirmed that translation efficiency was significantly reduced in cells overexpressing PepAA compared with the other model polypeptides evaluated. © 2005 Wiley Periodicals, Inc.</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>15736162</pmid><doi>10.1002/bit.20436</doi><tpages>11</tpages></addata></record> |
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| subjects | Amino Acid Sequence Amino acids Amino Acids - biosynthesis Amino Acids - chemistry Amino Acids - genetics Bacteria Biological and medical sciences Biotechnology Defensins - biosynthesis Defensins - chemistry Defensins - genetics Escherichia coli Escherichia coli - chemistry Escherichia coli - physiology Escherichia coli Proteins - biosynthesis Escherichia coli Proteins - chemistry Escherichia coli Proteins - genetics Fundamental and applied biological sciences. Psychology Gene expression Gene Expression Regulation, Bacterial - physiology Genetic engineering Genetic technics metabolic burden Metabolism Methods. Procedures. Technologies Modification of gene expression level Molecular Sequence Data Oxidative Stress - physiology Protein Engineering - methods Proteins recombinant protein production Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Structure-Activity Relationship transcriptional profiling translation efficiency |
| title | Amino acid content of recombinant proteins influences the metabolic burden response |
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