Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae
Recombinant human HbA, produced by co‐expressing α‐globin and β‐globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N‐terminal sequence analysis, peptide mapping, amino acid composition analysis and electr...
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| Veröffentlicht in: | European journal of biochemistry 1992-08, Vol.207 (3), p.931-936 |
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| container_title | European journal of biochemistry |
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| creator | COGHLAN, David JONES, Gareth DENTON, Katharine A. WILSON, Michael T. CHAN, Bernard HARRIS, Roy WOODROW, John R. OGDEN, Jill E. |
| description | Recombinant human HbA, produced by co‐expressing α‐globin and β‐globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N‐terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen‐binding studies demonstrated that the yeast‐derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb‐based oxygen carrier. |
| doi_str_mv | 10.1111/j.1432-1033.1992.tb17126.x |
| format | Article |
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Structural studies using N‐terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen‐binding studies demonstrated that the yeast‐derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb‐based oxygen carrier.</description><identifier>ISSN: 0014-2956</identifier><identifier>EISSN: 1432-1033</identifier><identifier>DOI: 10.1111/j.1432-1033.1992.tb17126.x</identifier><identifier>PMID: 1499566</identifier><identifier>CODEN: EJBCAI</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>Amino Acids - analysis ; Analytical, structural and metabolic biochemistry ; Biochemistry & Molecular Biology ; Biological and medical sciences ; Carbon Monoxide - metabolism ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel ; Fundamental and applied biological sciences. Psychology ; Hemoglobin A - genetics ; Hemoglobin A - metabolism ; Hemoproteins ; Humans ; Kinetics ; Life Sciences & Biomedicine ; Metalloproteins ; Peptide Mapping ; Plasmids ; Proteins ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae - genetics ; Science & Technology</subject><ispartof>European journal of biochemistry, 1992-08, Vol.207 (3), p.931-936</ispartof><rights>1992 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>true</woscitedreferencessubscribed><woscitedreferencescount>23</woscitedreferencescount><woscitedreferencesoriginalsourcerecordid>wosA1992JH20600013</woscitedreferencesoriginalsourcerecordid><citedby>FETCH-LOGICAL-c5181-64ec5563c456577c94ebc56028cf887581deba2b3361357b66a6b2151d72f7263</citedby><cites>FETCH-LOGICAL-c5181-64ec5563c456577c94ebc56028cf887581deba2b3361357b66a6b2151d72f7263</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,781,785,27197,27929,27930</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=5500004$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1499566$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>COGHLAN, David</creatorcontrib><creatorcontrib>JONES, Gareth</creatorcontrib><creatorcontrib>DENTON, Katharine A.</creatorcontrib><creatorcontrib>WILSON, Michael T.</creatorcontrib><creatorcontrib>CHAN, Bernard</creatorcontrib><creatorcontrib>HARRIS, Roy</creatorcontrib><creatorcontrib>WOODROW, John R.</creatorcontrib><creatorcontrib>OGDEN, Jill E.</creatorcontrib><title>Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae</title><title>European journal of biochemistry</title><addtitle>EUR J BIOCHEM</addtitle><addtitle>Eur J Biochem</addtitle><description>Recombinant human HbA, produced by co‐expressing α‐globin and β‐globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N‐terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen‐binding studies demonstrated that the yeast‐derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb‐based oxygen carrier.</description><subject>Amino Acids - analysis</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biochemistry & Molecular Biology</subject><subject>Biological and medical sciences</subject><subject>Carbon Monoxide - metabolism</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hemoglobin A - genetics</subject><subject>Hemoglobin A - metabolism</subject><subject>Hemoproteins</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Life Sciences & Biomedicine</subject><subject>Metalloproteins</subject><subject>Peptide Mapping</subject><subject>Plasmids</subject><subject>Proteins</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Saccharomyces cerevisiae</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Science & Technology</subject><issn>0014-2956</issn><issn>1432-1033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EZCTM</sourceid><sourceid>EIF</sourceid><recordid>eNqVkU2P0zAQhi0EWsrCT0CKEOKCEvwR2wkXVKpdFrQSh8LZcpwJdZXYxU6g_fc4NCpHwBdrZp53RjMvQi8ILkh6b_YFKRnNCWasIHVNi7EhklBRHB-g1aX0EK0wJmVOay4eoycx7jHGohbyCl2Rsk5ZsUJxO4bJjFPQfaZdm3WTM6P1LoVmp4M2IwQb9ZzKfJcFMH5orNNuzHbToF220zD4b71PyWydwfEQIEZosxRutZl7-OFkIGYGAvyw0Wp4ih51uo_wbPmv0dfbmy-bu_z-84ePm_V9bjipSC5KMJwLZkouuJSmLqExXGBama6qJK9IC42mDWOCMC4bIbRoKOGklbSTVLBr9Orc9xD89wniqAYbDfS9duCnqCQjuEz9_goSISSvK5zAt2fQBB9jgE4dgh10OCmC1WyN2qv5_mq-v5qtUYs16pjEz5cpUzNA-0d69iLVXy51HY3uu6CdsfGCcZ7sw2XCqjP2ExrfRWPBGbhQ63nopzuKRYIJ29jxt3cbP7kxSV__uzTR7xba9nD6j03V7c37bc0I-wVvks2g</recordid><startdate>199208</startdate><enddate>199208</enddate><creator>COGHLAN, David</creator><creator>JONES, Gareth</creator><creator>DENTON, Katharine A.</creator><creator>WILSON, Michael T.</creator><creator>CHAN, Bernard</creator><creator>HARRIS, Roy</creator><creator>WOODROW, John R.</creator><creator>OGDEN, Jill E.</creator><general>Blackwell Publishing Ltd</general><general>Springer Nature</general><general>Blackwell</general><scope>BLEPL</scope><scope>DTL</scope><scope>EZCTM</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>M7N</scope><scope>7X8</scope></search><sort><creationdate>199208</creationdate><title>Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae</title><author>COGHLAN, David ; JONES, Gareth ; DENTON, Katharine A. ; WILSON, Michael T. ; CHAN, Bernard ; HARRIS, Roy ; WOODROW, John R. ; OGDEN, Jill E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5181-64ec5563c456577c94ebc56028cf887581deba2b3361357b66a6b2151d72f7263</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Amino Acids - analysis</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biochemistry & Molecular Biology</topic><topic>Biological and medical sciences</topic><topic>Carbon Monoxide - metabolism</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hemoglobin A - genetics</topic><topic>Hemoglobin A - metabolism</topic><topic>Hemoproteins</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Life Sciences & Biomedicine</topic><topic>Metalloproteins</topic><topic>Peptide Mapping</topic><topic>Plasmids</topic><topic>Proteins</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Saccharomyces cerevisiae</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Science & Technology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>COGHLAN, David</creatorcontrib><creatorcontrib>JONES, Gareth</creatorcontrib><creatorcontrib>DENTON, Katharine A.</creatorcontrib><creatorcontrib>WILSON, Michael T.</creatorcontrib><creatorcontrib>CHAN, Bernard</creatorcontrib><creatorcontrib>HARRIS, Roy</creatorcontrib><creatorcontrib>WOODROW, John R.</creatorcontrib><creatorcontrib>OGDEN, Jill E.</creatorcontrib><collection>Web of Science Core Collection</collection><collection>Science Citation Index Expanded</collection><collection>Web of Science - Science Citation Index Expanded - 1992</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>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>MEDLINE - Academic</collection><jtitle>European journal of biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>COGHLAN, David</au><au>JONES, Gareth</au><au>DENTON, Katharine A.</au><au>WILSON, Michael T.</au><au>CHAN, Bernard</au><au>HARRIS, Roy</au><au>WOODROW, John R.</au><au>OGDEN, Jill E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae</atitle><jtitle>European journal of biochemistry</jtitle><stitle>EUR J BIOCHEM</stitle><addtitle>Eur J Biochem</addtitle><date>1992-08</date><risdate>1992</risdate><volume>207</volume><issue>3</issue><spage>931</spage><epage>936</epage><pages>931-936</pages><issn>0014-2956</issn><eissn>1432-1033</eissn><coden>EJBCAI</coden><abstract>Recombinant human HbA, produced by co‐expressing α‐globin and β‐globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N‐terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen‐binding studies demonstrated that the yeast‐derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb‐based oxygen carrier.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>1499566</pmid><doi>10.1111/j.1432-1033.1992.tb17126.x</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| source | Web of Science - Science Citation Index Expanded - 1992<img src="https://exlibris-pub.s3.amazonaws.com/fromwos-v2.jpg" />; MEDLINE; Alma/SFX Local Collection |
| subjects | Amino Acids - analysis Analytical, structural and metabolic biochemistry Biochemistry & Molecular Biology Biological and medical sciences Carbon Monoxide - metabolism Chromatography, High Pressure Liquid Electrophoresis, Polyacrylamide Gel Fundamental and applied biological sciences. Psychology Hemoglobin A - genetics Hemoglobin A - metabolism Hemoproteins Humans Kinetics Life Sciences & Biomedicine Metalloproteins Peptide Mapping Plasmids Proteins Recombinant Proteins - genetics Recombinant Proteins - metabolism Saccharomyces cerevisiae Saccharomyces cerevisiae - genetics Science & Technology |
| title | Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae |
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