Immunoaffinity purification of glucose/xylose isomerase from Streptomyces
A procedure was developed to purify glucose/xylose isomerase from cell extract of Streptomyces sp. NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of an...
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| Veröffentlicht in: | Applied biochemistry and biotechnology 1991-10, Vol.31 (1), p.11-20 |
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| container_title | Applied biochemistry and biotechnology |
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| creator | GHATGE, M MAWAL, Y GAIKWAD, D DESHPANDE, V |
| description | A procedure was developed to purify glucose/xylose isomerase from cell extract of Streptomyces sp. NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of antibodies was confirmed by double immunodiffusion, rocket electrophoresis, and Western-blot ELISA, which revealed the presence of a single immunoreactive protein with an Mr of 40,000. The antibodies recognized 2-3 antigenic determinants/mol of enzyme and were found to partially neutralize the enzymatic activity in an immunotitration experiment. The affinity gel was prepared by coupling antibodies at pH 10.0 to divinyl sulfone-activated Sepharose CL-4B. The glucose/xylose isomerase purified by immunoaffinity chromatography yielded 75% recovery with a single enzymatically active protein band on gel electrophoresis and showed specific activity of 16 U/mg. The crossreaction of the antibodies with glucose isomerase from other actinomycetes indicated that they share common epitopes. |
| doi_str_mv | 10.1007/BF02922121 |
| format | Article |
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NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of antibodies was confirmed by double immunodiffusion, rocket electrophoresis, and Western-blot ELISA, which revealed the presence of a single immunoreactive protein with an Mr of 40,000. The antibodies recognized 2-3 antigenic determinants/mol of enzyme and were found to partially neutralize the enzymatic activity in an immunotitration experiment. The affinity gel was prepared by coupling antibodies at pH 10.0 to divinyl sulfone-activated Sepharose CL-4B. The glucose/xylose isomerase purified by immunoaffinity chromatography yielded 75% recovery with a single enzymatically active protein band on gel electrophoresis and showed specific activity of 16 U/mg. The crossreaction of the antibodies with glucose isomerase from other actinomycetes indicated that they share common epitopes.</description><identifier>ISSN: 0273-2289</identifier><identifier>EISSN: 1559-0291</identifier><identifier>DOI: 10.1007/BF02922121</identifier><identifier>PMID: 1724596</identifier><identifier>CODEN: ABIBDL</identifier><language>eng</language><publisher>Heidelberg: Springer</publisher><subject>affinity chromatography ; Aldose-Ketose Isomerases ; Animals ; Antibody Specificity ; Biological and medical sciences ; Biotechnology ; Blotting, Western ; Carbohydrate Epimerases - immunology ; Carbohydrate Epimerases - isolation & purification ; Chromatography, Affinity - methods ; Cross Reactions ; Electrophoresis, Polyacrylamide Gel ; Enzyme engineering ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Fundamental and applied biological sciences. Psychology ; Immunodiffusion ; Improved methods for extraction and purification of enzymes ; Methods. Procedures. 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NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of antibodies was confirmed by double immunodiffusion, rocket electrophoresis, and Western-blot ELISA, which revealed the presence of a single immunoreactive protein with an Mr of 40,000. The antibodies recognized 2-3 antigenic determinants/mol of enzyme and were found to partially neutralize the enzymatic activity in an immunotitration experiment. The affinity gel was prepared by coupling antibodies at pH 10.0 to divinyl sulfone-activated Sepharose CL-4B. The glucose/xylose isomerase purified by immunoaffinity chromatography yielded 75% recovery with a single enzymatically active protein band on gel electrophoresis and showed specific activity of 16 U/mg. The crossreaction of the antibodies with glucose isomerase from other actinomycetes indicated that they share common epitopes.</description><subject>affinity chromatography</subject><subject>Aldose-Ketose Isomerases</subject><subject>Animals</subject><subject>Antibody Specificity</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Blotting, Western</subject><subject>Carbohydrate Epimerases - immunology</subject><subject>Carbohydrate Epimerases - isolation & purification</subject><subject>Chromatography, Affinity - methods</subject><subject>Cross Reactions</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzyme engineering</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>Epitopes</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Immunodiffusion</subject><subject>Improved methods for extraction and purification of enzymes</subject><subject>Methods. 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Technologies</subject><subject>polyclonal antibodies</subject><subject>Rabbits</subject><subject>Streptomyces - enzymology</subject><subject>xylose isomerase</subject><issn>0273-2289</issn><issn>1559-0291</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1991</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkM1LxDAQxYMo67p68S70IN7q5qNJmqMuri4seFDPZZomEmmamrRg_3srLl49vRnejze8QeiS4FuCsVzfbzFVlBJKjtCScK7yeSfHaImpZDmlpTpFZyl9YExoyeUCLYikBVdiiXY778cugLWuc8OU9WN01mkYXOiyYLP3dtQhmfXX1M6SuRS8iTBPNgafvQzR9EPwkzbpHJ1YaJO5OOgKvW0fXjdP-f75cbe52-c9FWLI68LImhhWK1WWJVjOmpryprG2UQSEBglEFwUTUBppTV2AmrtJo7iQCgxjK3Tzm9vH8DmaNFTeJW3aFjoTxlTJ-QxjlP8LEkGo4OoHvDqAY-1NU_XReYhTdXjS7F8ffEgaWhuh0y79YRxTIueYb8S4di8</recordid><startdate>19911001</startdate><enddate>19911001</enddate><creator>GHATGE, M</creator><creator>MAWAL, Y</creator><creator>GAIKWAD, D</creator><creator>DESHPANDE, V</creator><general>Springer</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QL</scope><scope>7QO</scope><scope>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M81</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>19911001</creationdate><title>Immunoaffinity purification of glucose/xylose isomerase from Streptomyces</title><author>GHATGE, M ; MAWAL, Y ; GAIKWAD, D ; DESHPANDE, V</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p266t-b4e7b1e3b99888af53db25ddffd91a6ca7a1c4436a8e7feb4a92927e95679ae33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1991</creationdate><topic>affinity chromatography</topic><topic>Aldose-Ketose Isomerases</topic><topic>Animals</topic><topic>Antibody Specificity</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Blotting, Western</topic><topic>Carbohydrate Epimerases - immunology</topic><topic>Carbohydrate Epimerases - isolation & purification</topic><topic>Chromatography, Affinity - methods</topic><topic>Cross Reactions</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzyme engineering</topic><topic>Enzyme-Linked Immunosorbent Assay</topic><topic>Epitopes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Immunodiffusion</topic><topic>Improved methods for extraction and purification of enzymes</topic><topic>Methods. Procedures. Technologies</topic><topic>polyclonal antibodies</topic><topic>Rabbits</topic><topic>Streptomyces - enzymology</topic><topic>xylose isomerase</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>GHATGE, M</creatorcontrib><creatorcontrib>MAWAL, Y</creatorcontrib><creatorcontrib>GAIKWAD, D</creatorcontrib><creatorcontrib>DESHPANDE, V</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>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>Biochemistry Abstracts 3</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Applied biochemistry and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>GHATGE, M</au><au>MAWAL, Y</au><au>GAIKWAD, D</au><au>DESHPANDE, V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Immunoaffinity purification of glucose/xylose isomerase from Streptomyces</atitle><jtitle>Applied biochemistry and biotechnology</jtitle><addtitle>Appl Biochem Biotechnol</addtitle><date>1991-10-01</date><risdate>1991</risdate><volume>31</volume><issue>1</issue><spage>11</spage><epage>20</epage><pages>11-20</pages><issn>0273-2289</issn><eissn>1559-0291</eissn><coden>ABIBDL</coden><abstract>A procedure was developed to purify glucose/xylose isomerase from cell extract of Streptomyces sp. NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of antibodies was confirmed by double immunodiffusion, rocket electrophoresis, and Western-blot ELISA, which revealed the presence of a single immunoreactive protein with an Mr of 40,000. The antibodies recognized 2-3 antigenic determinants/mol of enzyme and were found to partially neutralize the enzymatic activity in an immunotitration experiment. The affinity gel was prepared by coupling antibodies at pH 10.0 to divinyl sulfone-activated Sepharose CL-4B. The glucose/xylose isomerase purified by immunoaffinity chromatography yielded 75% recovery with a single enzymatically active protein band on gel electrophoresis and showed specific activity of 16 U/mg. 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| subjects | affinity chromatography Aldose-Ketose Isomerases Animals Antibody Specificity Biological and medical sciences Biotechnology Blotting, Western Carbohydrate Epimerases - immunology Carbohydrate Epimerases - isolation & purification Chromatography, Affinity - methods Cross Reactions Electrophoresis, Polyacrylamide Gel Enzyme engineering Enzyme-Linked Immunosorbent Assay Epitopes Fundamental and applied biological sciences. Psychology Immunodiffusion Improved methods for extraction and purification of enzymes Methods. Procedures. Technologies polyclonal antibodies Rabbits Streptomyces - enzymology xylose isomerase |
| title | Immunoaffinity purification of glucose/xylose isomerase from Streptomyces |
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