Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris
The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of post-translational modification. The expressed OVA con...
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| Veröffentlicht in: | Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2005-04, Vol.69 (4), p.755-761 |
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| creator | Ito, K. (Yamaguchi Univ. (Japan). Faculty of Agriculture) Matsudomi, N |
| description | The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of post-translational modification. The expressed OVA consisted of two molecular species immmuno-reactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosy-lated form respectively. The non-glycosylated form was not produced in the system. The other post-translational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tsub(m), which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 deg C as compared with that of hen egg OVA (77.3 deg C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermo-stability. |
| doi_str_mv | 10.1271/bbb.69.755 |
| format | Article |
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(Yamaguchi Univ. (Japan). Faculty of Agriculture) ; Matsudomi, N</creator><creatorcontrib>Ito, K. (Yamaguchi Univ. (Japan). Faculty of Agriculture) ; Matsudomi, N</creatorcontrib><description>The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of post-translational modification. The expressed OVA consisted of two molecular species immmuno-reactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosy-lated form respectively. The non-glycosylated form was not produced in the system. The other post-translational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tsub(m), which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 deg C as compared with that of hen egg OVA (77.3 deg C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermo-stability.</description><identifier>ISSN: 0916-8451</identifier><identifier>EISSN: 1347-6947</identifier><identifier>DOI: 10.1271/bbb.69.755</identifier><identifier>PMID: 15849414</identifier><language>eng</language><publisher>Tokyo: Japan Society for Bioscience, Biotechnology, and Agrochemistry</publisher><subject>ACETYLATION ; ALBUMINS ; Amino Acid Sequence ; AMINO ACID SEQUENCES ; Animals ; Biological and medical sciences ; BIOTECHNOLOGY ; Chickens ; Circular Dichroism ; EGG WHITE ; Fundamental and applied biological sciences. Psychology ; GENE EXPRESSION ; Genetic Vectors - genetics ; Glycosylation ; Mass Spectrometry ; N-linked glycosylation ; ovalbumin ; Ovalbumin - biosynthesis ; Ovalbumin - chemistry ; Ovalbumin - genetics ; Ovalbumin - isolation & purification ; Ovum ; PHOSPHORYLATION ; Pichia - genetics ; Pichia - metabolism ; PICHIA PASTORIS ; Pichia pastoris expression ; Protein Denaturation ; Protein Folding ; RECOMBINANT PROTEINS ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Spectrometry, Fluorescence ; Temperature ; TRYPTOPHAN</subject><ispartof>Bioscience, biotechnology, and biochemistry, 2005-04, Vol.69 (4), p.755-761</ispartof><rights>2005 by Japan Society for Bioscience, Biotechnology, and Agrochemistry 2005</rights><rights>2005 INIST-CNRS</rights><rights>Copyright Japan Science and Technology Agency 2005</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c537t-1da42e3e5dc65358f153bf0da15613bc6262f03e3d0c8e5d2d177a7a0ddc0b623</citedby><cites>FETCH-LOGICAL-c537t-1da42e3e5dc65358f153bf0da15613bc6262f03e3d0c8e5d2d177a7a0ddc0b623</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=16903740$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15849414$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ito, K. (Yamaguchi Univ. (Japan). Faculty of Agriculture)</creatorcontrib><creatorcontrib>Matsudomi, N</creatorcontrib><title>Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris</title><title>Bioscience, biotechnology, and biochemistry</title><addtitle>Biosci Biotechnol Biochem</addtitle><description>The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of post-translational modification. The expressed OVA consisted of two molecular species immmuno-reactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosy-lated form respectively. The non-glycosylated form was not produced in the system. The other post-translational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tsub(m), which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 deg C as compared with that of hen egg OVA (77.3 deg C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermo-stability.</description><subject>ACETYLATION</subject><subject>ALBUMINS</subject><subject>Amino Acid Sequence</subject><subject>AMINO ACID SEQUENCES</subject><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>BIOTECHNOLOGY</subject><subject>Chickens</subject><subject>Circular Dichroism</subject><subject>EGG WHITE</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>GENE EXPRESSION</subject><subject>Genetic Vectors - genetics</subject><subject>Glycosylation</subject><subject>Mass Spectrometry</subject><subject>N-linked glycosylation</subject><subject>ovalbumin</subject><subject>Ovalbumin - biosynthesis</subject><subject>Ovalbumin - chemistry</subject><subject>Ovalbumin - genetics</subject><subject>Ovalbumin - isolation & purification</subject><subject>Ovum</subject><subject>PHOSPHORYLATION</subject><subject>Pichia - genetics</subject><subject>Pichia - metabolism</subject><subject>PICHIA PASTORIS</subject><subject>Pichia pastoris expression</subject><subject>Protein Denaturation</subject><subject>Protein Folding</subject><subject>RECOMBINANT PROTEINS</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Spectrometry, Fluorescence</subject><subject>Temperature</subject><subject>TRYPTOPHAN</subject><issn>0916-8451</issn><issn>1347-6947</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpt0MtrFEEQB-BGFLOJXrwrA6IHYdau6dfMUYIxhoDBx7mp6cduh5nptXtG3f8-LbshIJ66q_iqKH6EvAC6hkbB-77v17JbKyEekRUwrmrZcfWYrGgHsm65gBNymvMtpaUh4Ck5AdHyjgNfka_f5rSYeUk4VGaLCc3sUshzMLmKvtq6qXKbTRV_4dAvYyjVn11yOTtblWLvMM_VTTDbgNWu_GOZfUaeeByye358z8iPi4_fzy_r6y-fPp9_uK6NYGquwSJvHHPCGimYaD0I1ntqEYQE1hvZyMZT5pilpi2qsaAUKqTWGtrLhp2Rt4e9uxR_Li7PegzZuGHAycUla6mUAk7bAl__A2_jkqZymwbOuxZAdrKodwdlUsw5Oa93KYyY9hqo_puzLjlr2emSc8GvjiuXfnT2gR6DLeDNEWA2OPiEkwn5wcmOMsVpceLgwuRjGvF3TIPVM-6HmO6H2H8PeHmY8xg1bkrs-uqmoVRQqloB7A7RBKCj</recordid><startdate>20050401</startdate><enddate>20050401</enddate><creator>Ito, K. (Yamaguchi Univ. (Japan). Faculty of Agriculture)</creator><creator>Matsudomi, N</creator><general>Japan Society for Bioscience, Biotechnology, and Agrochemistry</general><general>Japan Society for Bioscience Biotechnology and Agrochemistry</general><general>Oxford University Press</general><scope>FBQ</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>7X8</scope></search><sort><creationdate>20050401</creationdate><title>Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris</title><author>Ito, K. (Yamaguchi Univ. (Japan). Faculty of Agriculture) ; Matsudomi, N</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c537t-1da42e3e5dc65358f153bf0da15613bc6262f03e3d0c8e5d2d177a7a0ddc0b623</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>ACETYLATION</topic><topic>ALBUMINS</topic><topic>Amino Acid Sequence</topic><topic>AMINO ACID SEQUENCES</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>BIOTECHNOLOGY</topic><topic>Chickens</topic><topic>Circular Dichroism</topic><topic>EGG WHITE</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>GENE EXPRESSION</topic><topic>Genetic Vectors - genetics</topic><topic>Glycosylation</topic><topic>Mass Spectrometry</topic><topic>N-linked glycosylation</topic><topic>ovalbumin</topic><topic>Ovalbumin - biosynthesis</topic><topic>Ovalbumin - chemistry</topic><topic>Ovalbumin - genetics</topic><topic>Ovalbumin - isolation & purification</topic><topic>Ovum</topic><topic>PHOSPHORYLATION</topic><topic>Pichia - genetics</topic><topic>Pichia - metabolism</topic><topic>PICHIA PASTORIS</topic><topic>Pichia pastoris expression</topic><topic>Protein Denaturation</topic><topic>Protein Folding</topic><topic>RECOMBINANT PROTEINS</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Spectrometry, Fluorescence</topic><topic>Temperature</topic><topic>TRYPTOPHAN</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ito, K. (Yamaguchi Univ. (Japan). Faculty of Agriculture)</creatorcontrib><creatorcontrib>Matsudomi, N</creatorcontrib><collection>AGRIS</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>MEDLINE - Academic</collection><jtitle>Bioscience, biotechnology, and biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ito, K. (Yamaguchi Univ. (Japan). Faculty of Agriculture)</au><au>Matsudomi, N</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris</atitle><jtitle>Bioscience, biotechnology, and biochemistry</jtitle><addtitle>Biosci Biotechnol Biochem</addtitle><date>2005-04-01</date><risdate>2005</risdate><volume>69</volume><issue>4</issue><spage>755</spage><epage>761</epage><pages>755-761</pages><issn>0916-8451</issn><eissn>1347-6947</eissn><abstract>The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of post-translational modification. The expressed OVA consisted of two molecular species immmuno-reactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosy-lated form respectively. The non-glycosylated form was not produced in the system. The other post-translational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tsub(m), which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 deg C as compared with that of hen egg OVA (77.3 deg C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermo-stability.</abstract><cop>Tokyo</cop><pub>Japan Society for Bioscience, Biotechnology, and Agrochemistry</pub><pmid>15849414</pmid><doi>10.1271/bbb.69.755</doi><tpages>7</tpages></addata></record> |
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| source | J-STAGE Free; Oxford University Press Journals All Titles (1996-Current); MEDLINE; Freely Accessible Japanese Titles; EZB-FREE-00999 freely available EZB journals; Free Full-Text Journals in Chemistry |
| subjects | ACETYLATION ALBUMINS Amino Acid Sequence AMINO ACID SEQUENCES Animals Biological and medical sciences BIOTECHNOLOGY Chickens Circular Dichroism EGG WHITE Fundamental and applied biological sciences. Psychology GENE EXPRESSION Genetic Vectors - genetics Glycosylation Mass Spectrometry N-linked glycosylation ovalbumin Ovalbumin - biosynthesis Ovalbumin - chemistry Ovalbumin - genetics Ovalbumin - isolation & purification Ovum PHOSPHORYLATION Pichia - genetics Pichia - metabolism PICHIA PASTORIS Pichia pastoris expression Protein Denaturation Protein Folding RECOMBINANT PROTEINS Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Spectrometry, Fluorescence Temperature TRYPTOPHAN |
| title | Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris |
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