Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities
The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the s...
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| Veröffentlicht in: | Proteomics (Weinheim) 2005-12, Vol.5 (18), p.4689-4704 |
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| creator | Alete, Daniel E. Racher, Andrew J. Birch, John R. Stansfield, Scott H. James, David C. Smales, C. Mark |
| description | The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high‐level antibody secretion. We used GS‐NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid‐exponential growth phase. These were analysed by 2‐D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up‐regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell‐engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study. |
| doi_str_mv | 10.1002/pmic.200500019 |
| format | Article |
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Mark</creator><creatorcontrib>Alete, Daniel E. ; Racher, Andrew J. ; Birch, John R. ; Stansfield, Scott H. ; James, David C. ; Smales, C. Mark</creatorcontrib><description>The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high‐level antibody secretion. We used GS‐NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid‐exponential growth phase. These were analysed by 2‐D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up‐regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell‐engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study.</description><identifier>ISSN: 1615-9853</identifier><identifier>EISSN: 1615-9861</identifier><identifier>DOI: 10.1002/pmic.200500019</identifier><identifier>PMID: 16247733</identifier><language>eng</language><publisher>Weinheim: WILEY-VCH Verlag</publisher><subject>Analytical, structural and metabolic biochemistry ; Animals ; Antibodies, Monoclonal - biosynthesis ; Antibodies, Monoclonal - genetics ; Applied microbiology ; Biological and medical sciences ; Cell Line, Tumor ; Cell specific productivity ; Electrophoresis, Gel, Two-Dimensional ; Enriched microsome fraction ; Fundamental and applied biological sciences. Psychology ; Gene Expression Profiling - methods ; Gene Expression Regulation, Neoplastic ; Glutamate-Ammonia Ligase - biosynthesis ; Glutamate-Ammonia Ligase - genetics ; Hematologic and hematopoietic diseases ; Immunodeficiencies. Immunoglobulinopathies ; Immunoglobulinopathies ; Immunopathology ; Leukemias. Malignant lymphomas. Malignant reticulosis. Myelofibrosis ; Medical sciences ; Mice ; Microbiology ; Microsomes - metabolism ; Miscellaneous ; Monoclonal antibody production ; Multiple Myeloma - metabolism ; NS0 murine myeloma cells ; Proteins ; Proteome - metabolism ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - genetics ; Secretory pathway ; Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies (general aspects)</subject><ispartof>Proteomics (Weinheim), 2005-12, Vol.5 (18), p.4689-4704</ispartof><rights>Copyright © 2005 WILEY‐VCH Verlag GmbH & Co. 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Mark</creatorcontrib><title>Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities</title><title>Proteomics (Weinheim)</title><addtitle>Proteomics</addtitle><description>The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high‐level antibody secretion. We used GS‐NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid‐exponential growth phase. These were analysed by 2‐D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up‐regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell‐engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Antibodies, Monoclonal - biosynthesis</subject><subject>Antibodies, Monoclonal - genetics</subject><subject>Applied microbiology</subject><subject>Biological and medical sciences</subject><subject>Cell Line, Tumor</subject><subject>Cell specific productivity</subject><subject>Electrophoresis, Gel, Two-Dimensional</subject><subject>Enriched microsome fraction</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression Profiling - methods</subject><subject>Gene Expression Regulation, Neoplastic</subject><subject>Glutamate-Ammonia Ligase - biosynthesis</subject><subject>Glutamate-Ammonia Ligase - genetics</subject><subject>Hematologic and hematopoietic diseases</subject><subject>Immunodeficiencies. Immunoglobulinopathies</subject><subject>Immunoglobulinopathies</subject><subject>Immunopathology</subject><subject>Leukemias. Malignant lymphomas. Malignant reticulosis. Myelofibrosis</subject><subject>Medical sciences</subject><subject>Mice</subject><subject>Microbiology</subject><subject>Microsomes - metabolism</subject><subject>Miscellaneous</subject><subject>Monoclonal antibody production</subject><subject>Multiple Myeloma - metabolism</subject><subject>NS0 murine myeloma cells</subject><subject>Proteins</subject><subject>Proteome - metabolism</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - genetics</subject><subject>Secretory pathway</subject><subject>Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies (general aspects)</subject><issn>1615-9853</issn><issn>1615-9861</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1u1DAUhSMEoqWwZYm8gV0GO07iZIlGMBS1Q0UHsbQc-5oa_DO1k5Y8Cm-LRzOasuvKR_J3z7HvKYrXBC8IxtX7rTNyUWHcYIxJ_6Q4JS1pyr5rydOjbuhJ8SKlX5lgXc-eFyekrWrGKD0t_l7FMELILkh4YedkEgoagY9G3oBC-SKGFJywSEchRxN8yio4tLou19cYuSkaD8jNYDOFJFib0L0Zb9CdiLPxP1ECGWHMXhFkcIPxwo_IBR-kDTky545mCGpG2xjUlCPuzGggvSyeaWETvDqcZ8X3Tx83y8_lxdfV-fLDRSkpq_pSUT0ArZUclNRZdFLXQBqosGiF1JoqSoZh6EBK1fZDI2pcKawoNFgrqWp6Vrzb--b42wnSyJ1Ju28ID2FKvO163HYVfhQkrGYtrVgGF3twt7oUQfNtNC5vgxPMd63xXWv82FoeeHNwngYH6gE_1JSBtwdAJClsbsJLkx64zNQ5OHP9nrs3FuZHYvnV5fny_0eU-1mTRvhznBXxN28ZZQ3_sV7xyy_f1ptmueGY_gNxi8ZT</recordid><startdate>20051201</startdate><enddate>20051201</enddate><creator>Alete, Daniel E.</creator><creator>Racher, Andrew J.</creator><creator>Birch, John R.</creator><creator>Stansfield, Scott H.</creator><creator>James, David C.</creator><creator>Smales, C. Mark</creator><general>WILEY-VCH Verlag</general><general>WILEY‐VCH Verlag</general><general>Wiley-VCH</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>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20051201</creationdate><title>Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities</title><author>Alete, Daniel E. ; Racher, Andrew J. ; Birch, John R. ; Stansfield, Scott H. ; James, David C. ; Smales, C. Mark</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3729-d3fbe34dcbdcfe348cf4e15e20a6acff3d31bbb8eccd69b5a402d0d3e50fdcd43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Antibodies, Monoclonal - biosynthesis</topic><topic>Antibodies, Monoclonal - genetics</topic><topic>Applied microbiology</topic><topic>Biological and medical sciences</topic><topic>Cell Line, Tumor</topic><topic>Cell specific productivity</topic><topic>Electrophoresis, Gel, Two-Dimensional</topic><topic>Enriched microsome fraction</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression Profiling - methods</topic><topic>Gene Expression Regulation, Neoplastic</topic><topic>Glutamate-Ammonia Ligase - biosynthesis</topic><topic>Glutamate-Ammonia Ligase - genetics</topic><topic>Hematologic and hematopoietic diseases</topic><topic>Immunodeficiencies. Immunoglobulinopathies</topic><topic>Immunoglobulinopathies</topic><topic>Immunopathology</topic><topic>Leukemias. Malignant lymphomas. Malignant reticulosis. Myelofibrosis</topic><topic>Medical sciences</topic><topic>Mice</topic><topic>Microbiology</topic><topic>Microsomes - metabolism</topic><topic>Miscellaneous</topic><topic>Monoclonal antibody production</topic><topic>Multiple Myeloma - metabolism</topic><topic>NS0 murine myeloma cells</topic><topic>Proteins</topic><topic>Proteome - metabolism</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - genetics</topic><topic>Secretory pathway</topic><topic>Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies (general aspects)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Alete, Daniel E.</creatorcontrib><creatorcontrib>Racher, Andrew J.</creatorcontrib><creatorcontrib>Birch, John R.</creatorcontrib><creatorcontrib>Stansfield, Scott H.</creatorcontrib><creatorcontrib>James, David C.</creatorcontrib><creatorcontrib>Smales, C. 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Mark</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities</atitle><jtitle>Proteomics (Weinheim)</jtitle><addtitle>Proteomics</addtitle><date>2005-12-01</date><risdate>2005</risdate><volume>5</volume><issue>18</issue><spage>4689</spage><epage>4704</epage><pages>4689-4704</pages><issn>1615-9853</issn><eissn>1615-9861</eissn><abstract>The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high‐level antibody secretion. We used GS‐NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid‐exponential growth phase. These were analysed by 2‐D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up‐regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell‐engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study.</abstract><cop>Weinheim</cop><pub>WILEY-VCH Verlag</pub><pmid>16247733</pmid><doi>10.1002/pmic.200500019</doi><tpages>16</tpages></addata></record> |
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| subjects | Analytical, structural and metabolic biochemistry Animals Antibodies, Monoclonal - biosynthesis Antibodies, Monoclonal - genetics Applied microbiology Biological and medical sciences Cell Line, Tumor Cell specific productivity Electrophoresis, Gel, Two-Dimensional Enriched microsome fraction Fundamental and applied biological sciences. Psychology Gene Expression Profiling - methods Gene Expression Regulation, Neoplastic Glutamate-Ammonia Ligase - biosynthesis Glutamate-Ammonia Ligase - genetics Hematologic and hematopoietic diseases Immunodeficiencies. Immunoglobulinopathies Immunoglobulinopathies Immunopathology Leukemias. Malignant lymphomas. Malignant reticulosis. Myelofibrosis Medical sciences Mice Microbiology Microsomes - metabolism Miscellaneous Monoclonal antibody production Multiple Myeloma - metabolism NS0 murine myeloma cells Proteins Proteome - metabolism Recombinant Proteins - biosynthesis Recombinant Proteins - genetics Secretory pathway Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies (general aspects) |
| title | Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities |
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