High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells
We have developed a high-expression system of recombinant human mannan-binding lectin (MBL) with CHO cells. Geneticin-resistant transformants harboring human MBL cDNA in the expression vector pNOW/CMV-A were screened by immunoblot analysis for secretion of recombinant MBL. Cloning and selection by b...
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| Veröffentlicht in: | Journal of immunological methods 1999, Vol.222 (1), p.135-144 |
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| creator | Ohtani, Katsuki Suzuki, Yasuhiko Eda, Souji Kawai, Takao Kase, Tetsuo Keshi, Hiroyuki Sakai, Yoshinori Yamamoto, Satoshi Sakamoto, Takashi Wakamiya, Nobutaka |
| description | We have developed a high-expression system of recombinant human mannan-binding lectin (MBL) with CHO cells. Geneticin-resistant transformants harboring human MBL cDNA in the expression vector pNOW/CMV-A were screened by immunoblot analysis for secretion of recombinant MBL. Cloning and selection by both geneticin and methotrexate resulted in the production of recombinant MBL to a final concentration of 128.8 μg/ml in media after four days of culture. SDS-PAGE and gel-filtration analyses showed that recombinant MBL is characterized by two lower-order oligomeric structures (apparent molecular weights: 1150 kDa and 300 kDa) compared to native MBL (apparent molecular weight: 1300 kDa). The recombinant human MBL has both sugar-binding and complement activation activity and, like native MBL, can inhibit hemagglutination of influenza A virus. Lectin blots with recombinant MBL indicate that it can bind such microorganisms as HIV and influenza virus suggesting that it might inhibit their infection of hosts. This high-level expression of human MBL with the full range of biological activity will be useful for studies on the immunological role of MBL in humans. |
| doi_str_mv | 10.1016/S0022-1759(98)00190-2 |
| format | Article |
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Geneticin-resistant transformants harboring human MBL cDNA in the expression vector pNOW/CMV-A were screened by immunoblot analysis for secretion of recombinant MBL. Cloning and selection by both geneticin and methotrexate resulted in the production of recombinant MBL to a final concentration of 128.8 μg/ml in media after four days of culture. SDS-PAGE and gel-filtration analyses showed that recombinant MBL is characterized by two lower-order oligomeric structures (apparent molecular weights: 1150 kDa and 300 kDa) compared to native MBL (apparent molecular weight: 1300 kDa). The recombinant human MBL has both sugar-binding and complement activation activity and, like native MBL, can inhibit hemagglutination of influenza A virus. Lectin blots with recombinant MBL indicate that it can bind such microorganisms as HIV and influenza virus suggesting that it might inhibit their infection of hosts. This high-level expression of human MBL with the full range of biological activity will be useful for studies on the immunological role of MBL in humans.</description><identifier>ISSN: 0022-1759</identifier><identifier>EISSN: 1872-7905</identifier><identifier>DOI: 10.1016/S0022-1759(98)00190-2</identifier><identifier>PMID: 10022380</identifier><identifier>CODEN: JIMMBG</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Animals ; Anti-viral activity ; Biological and medical sciences ; Biotechnology ; Carrier Proteins - biosynthesis ; Carrier Proteins - genetics ; Carrier Proteins - isolation & purification ; CHO Cells - metabolism ; CHO expression system ; Collectins ; Complement activation ; Cricetinae ; DNA, Complementary - genetics ; DNA, Complementary - metabolism ; Electrophoresis, Polyacrylamide Gel ; Fundamental and applied biological sciences. Psychology ; Gene Amplification ; Hemagglutination Inhibition Tests ; Humans ; Mannan-binding lectin (MBL) ; Methods. Procedures. Technologies ; Protein engineering ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Transformation, Genetic</subject><ispartof>Journal of immunological methods, 1999, Vol.222 (1), p.135-144</ispartof><rights>1999 Elsevier Science B.V.</rights><rights>1999 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c487t-dbb9d565592f3a09ad6bb34ada7ac37988e4001d24dd745cd7972bfc3e3069d53</citedby><cites>FETCH-LOGICAL-c487t-dbb9d565592f3a09ad6bb34ada7ac37988e4001d24dd745cd7972bfc3e3069d53</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0022175998001902$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,4010,27902,27903,27904,65309</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1660532$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10022380$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ohtani, Katsuki</creatorcontrib><creatorcontrib>Suzuki, Yasuhiko</creatorcontrib><creatorcontrib>Eda, Souji</creatorcontrib><creatorcontrib>Kawai, Takao</creatorcontrib><creatorcontrib>Kase, Tetsuo</creatorcontrib><creatorcontrib>Keshi, Hiroyuki</creatorcontrib><creatorcontrib>Sakai, Yoshinori</creatorcontrib><creatorcontrib>Yamamoto, Satoshi</creatorcontrib><creatorcontrib>Sakamoto, Takashi</creatorcontrib><creatorcontrib>Wakamiya, Nobutaka</creatorcontrib><title>High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells</title><title>Journal of immunological methods</title><addtitle>J Immunol Methods</addtitle><description>We have developed a high-expression system of recombinant human mannan-binding lectin (MBL) with CHO cells. Geneticin-resistant transformants harboring human MBL cDNA in the expression vector pNOW/CMV-A were screened by immunoblot analysis for secretion of recombinant MBL. Cloning and selection by both geneticin and methotrexate resulted in the production of recombinant MBL to a final concentration of 128.8 μg/ml in media after four days of culture. SDS-PAGE and gel-filtration analyses showed that recombinant MBL is characterized by two lower-order oligomeric structures (apparent molecular weights: 1150 kDa and 300 kDa) compared to native MBL (apparent molecular weight: 1300 kDa). The recombinant human MBL has both sugar-binding and complement activation activity and, like native MBL, can inhibit hemagglutination of influenza A virus. Lectin blots with recombinant MBL indicate that it can bind such microorganisms as HIV and influenza virus suggesting that it might inhibit their infection of hosts. This high-level expression of human MBL with the full range of biological activity will be useful for studies on the immunological role of MBL in humans.</description><subject>Animals</subject><subject>Anti-viral activity</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Carrier Proteins - biosynthesis</subject><subject>Carrier Proteins - genetics</subject><subject>Carrier Proteins - isolation & purification</subject><subject>CHO Cells - metabolism</subject><subject>CHO expression system</subject><subject>Collectins</subject><subject>Complement activation</subject><subject>Cricetinae</subject><subject>DNA, Complementary - genetics</subject><subject>DNA, Complementary - metabolism</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Amplification</subject><subject>Hemagglutination Inhibition Tests</subject><subject>Humans</subject><subject>Mannan-binding lectin (MBL)</subject><subject>Methods. Procedures. Technologies</subject><subject>Protein engineering</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Transformation, Genetic</subject><issn>0022-1759</issn><issn>1872-7905</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtvEzEUhS1ERdPCTwB5gVCymOLH-LWqICqkUlAXwNry2HcaoxlPGWci8e_xNBF014XlK_k71_fcg9BbSq4oofLjd0IYq6gSZmn0ihBqSMVeoAXVilXKEPESLf4h5-gi51-kUESSV-iczi9ckwXqN_F-V3VwgA67FDC0Lfh9PAB-GIcwlXJIeGjxbupdwuUkl6omphDTPe5mNOHlt8_bFS7FehcTZMA71-c9jHg4uPEPXq43dyvsoevya3TWui7Dm9N9iX5-ufmx3lTbu6-360_bytda7avQNCYIKYRhLXfEuCCbhtcuOOU8V0ZrqIuXwOoQVC18UEaxpvUcOJFFyS_Rh2PfYuL3BHlv-5jnCVyCYcpWGqGM1PpZkCrKGReqgOII-nHIeYTWPoyxL_YsJXYOxD4GYudtW6PtYyCWFd270wdT00N4ojomUID3J8Bl77p2dMnH_J-Tkgg-97k-YlDWdogw2uwjJA8hjiUFG4b4zCR_AZUZpl4</recordid><startdate>1999</startdate><enddate>1999</enddate><creator>Ohtani, Katsuki</creator><creator>Suzuki, Yasuhiko</creator><creator>Eda, Souji</creator><creator>Kawai, Takao</creator><creator>Kase, Tetsuo</creator><creator>Keshi, Hiroyuki</creator><creator>Sakai, Yoshinori</creator><creator>Yamamoto, Satoshi</creator><creator>Sakamoto, Takashi</creator><creator>Wakamiya, Nobutaka</creator><general>Elsevier B.V</general><general>Elsevier</general><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>7T5</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>1999</creationdate><title>High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells</title><author>Ohtani, Katsuki ; Suzuki, Yasuhiko ; Eda, Souji ; Kawai, Takao ; Kase, Tetsuo ; Keshi, Hiroyuki ; Sakai, Yoshinori ; Yamamoto, Satoshi ; Sakamoto, Takashi ; Wakamiya, Nobutaka</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c487t-dbb9d565592f3a09ad6bb34ada7ac37988e4001d24dd745cd7972bfc3e3069d53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Animals</topic><topic>Anti-viral activity</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Carrier Proteins - biosynthesis</topic><topic>Carrier Proteins - genetics</topic><topic>Carrier Proteins - isolation & purification</topic><topic>CHO Cells - metabolism</topic><topic>CHO expression system</topic><topic>Collectins</topic><topic>Complement activation</topic><topic>Cricetinae</topic><topic>DNA, Complementary - genetics</topic><topic>DNA, Complementary - metabolism</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Amplification</topic><topic>Hemagglutination Inhibition Tests</topic><topic>Humans</topic><topic>Mannan-binding lectin (MBL)</topic><topic>Methods. Procedures. Technologies</topic><topic>Protein engineering</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Transformation, Genetic</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ohtani, Katsuki</creatorcontrib><creatorcontrib>Suzuki, Yasuhiko</creatorcontrib><creatorcontrib>Eda, Souji</creatorcontrib><creatorcontrib>Kawai, Takao</creatorcontrib><creatorcontrib>Kase, Tetsuo</creatorcontrib><creatorcontrib>Keshi, Hiroyuki</creatorcontrib><creatorcontrib>Sakai, Yoshinori</creatorcontrib><creatorcontrib>Yamamoto, Satoshi</creatorcontrib><creatorcontrib>Sakamoto, Takashi</creatorcontrib><creatorcontrib>Wakamiya, Nobutaka</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>Biotechnology Research Abstracts</collection><collection>Immunology Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of immunological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ohtani, Katsuki</au><au>Suzuki, Yasuhiko</au><au>Eda, Souji</au><au>Kawai, Takao</au><au>Kase, Tetsuo</au><au>Keshi, Hiroyuki</au><au>Sakai, Yoshinori</au><au>Yamamoto, Satoshi</au><au>Sakamoto, Takashi</au><au>Wakamiya, Nobutaka</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells</atitle><jtitle>Journal of immunological methods</jtitle><addtitle>J Immunol Methods</addtitle><date>1999</date><risdate>1999</risdate><volume>222</volume><issue>1</issue><spage>135</spage><epage>144</epage><pages>135-144</pages><issn>0022-1759</issn><eissn>1872-7905</eissn><coden>JIMMBG</coden><abstract>We have developed a high-expression system of recombinant human mannan-binding lectin (MBL) with CHO cells. Geneticin-resistant transformants harboring human MBL cDNA in the expression vector pNOW/CMV-A were screened by immunoblot analysis for secretion of recombinant MBL. Cloning and selection by both geneticin and methotrexate resulted in the production of recombinant MBL to a final concentration of 128.8 μg/ml in media after four days of culture. SDS-PAGE and gel-filtration analyses showed that recombinant MBL is characterized by two lower-order oligomeric structures (apparent molecular weights: 1150 kDa and 300 kDa) compared to native MBL (apparent molecular weight: 1300 kDa). The recombinant human MBL has both sugar-binding and complement activation activity and, like native MBL, can inhibit hemagglutination of influenza A virus. Lectin blots with recombinant MBL indicate that it can bind such microorganisms as HIV and influenza virus suggesting that it might inhibit their infection of hosts. This high-level expression of human MBL with the full range of biological activity will be useful for studies on the immunological role of MBL in humans.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>10022380</pmid><doi>10.1016/S0022-1759(98)00190-2</doi><tpages>10</tpages></addata></record> |
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| ispartof | Journal of immunological methods, 1999, Vol.222 (1), p.135-144 |
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| subjects | Animals Anti-viral activity Biological and medical sciences Biotechnology Carrier Proteins - biosynthesis Carrier Proteins - genetics Carrier Proteins - isolation & purification CHO Cells - metabolism CHO expression system Collectins Complement activation Cricetinae DNA, Complementary - genetics DNA, Complementary - metabolism Electrophoresis, Polyacrylamide Gel Fundamental and applied biological sciences. Psychology Gene Amplification Hemagglutination Inhibition Tests Humans Mannan-binding lectin (MBL) Methods. Procedures. Technologies Protein engineering Recombinant Proteins - biosynthesis Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Transformation, Genetic |
| title | High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells |
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