Biological activity, membrane‐targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli
Decay‐accelerating factor (DAF, CD55) is a glycophosphatidyl inositol‐anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeut...
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| Veröffentlicht in: | Protein science 2004-09, Vol.13 (9), p.2406-2415 |
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| creator | White, Jennifer Lukacik, Petra Esser, Dirk Steward, Michael Giddings, Naomi Bright, Jeremy R. Fritchley, Sarah J. Morgan, B. Paul Lea, Susan M. Smith, Geoffrey P. Smith, Richard A.G. |
| description | Decay‐accelerating factor (DAF, CD55) is a glycophosphatidyl inositol‐anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane‐targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C‐terminal cysteine residue to permit site‐specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane‐localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF‐derived therapeutic. Crystals of the E. coli‐derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X‐ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential. |
| doi_str_mv | 10.1110/ps.03455604 |
| format | Article |
| fullrecord | <record><control><sourceid>wiley_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2280017</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>PRO132406</sourcerecordid><originalsourceid>FETCH-LOGICAL-c3836-63ae3fb0d1c61d6c712f18541c0a02405ff496872ec8c6cf1584194c4eea767a3</originalsourceid><addsrcrecordid>eNp9kU1v1DAQhq0K1G4LJ-7Id5rFEztOckGiVVuQKrWqQOJmeZ3x1siJIztbCCfuvfQ39pdguuXrwsnS-JlnNPMS8gLYEgDY6zEtGRdVJZnYIQsQsi2aVn56QhaslVA0XDZ7ZD-lz4wxASXfJXtQ8bIsG74gt0cu-LB2RnuqzeRu3DQf0h77VdQD3n-_m3Rc4-SGNe1D52wGJxeGQ6qHjpo4p0l77749FGmwNAW_WXmk15teD7RDo-fsNegx6geLzVNCpPh1jJgSdtQN9GRJTfDuGXlqtU_4_PE9IB9PTz4cvyvOL87eH789LwzPyxSSa-R2xTowEjppaigtNJUAwzQrBausFa1s6hJNY6SxUDUCWmEEoq5lrfkBebP1jptVj53BYYraqzG6XsdZBe3Uvz-Du1brcKPyyRiDOgtebQUmhpQi2t-9wNTPTNSY1K9MMv3y73F_2McQMlBugS_O4_w_l7q8ugCed5T8B8Tfm88</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype></control><display><type>article</type><title>Biological activity, membrane‐targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli</title><source>Wiley Free Content</source><source>MEDLINE</source><source>Wiley Online Library Journals Frontfile Complete</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>PubMed Central</source><source>Free Full-Text Journals in Chemistry</source><creator>White, Jennifer ; Lukacik, Petra ; Esser, Dirk ; Steward, Michael ; Giddings, Naomi ; Bright, Jeremy R. ; Fritchley, Sarah J. ; Morgan, B. Paul ; Lea, Susan M. ; Smith, Geoffrey P. ; Smith, Richard A.G.</creator><creatorcontrib>White, Jennifer ; Lukacik, Petra ; Esser, Dirk ; Steward, Michael ; Giddings, Naomi ; Bright, Jeremy R. ; Fritchley, Sarah J. ; Morgan, B. Paul ; Lea, Susan M. ; Smith, Geoffrey P. ; Smith, Richard A.G.</creatorcontrib><description>Decay‐accelerating factor (DAF, CD55) is a glycophosphatidyl inositol‐anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane‐targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C‐terminal cysteine residue to permit site‐specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane‐localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF‐derived therapeutic. Crystals of the E. coli‐derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X‐ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.</description><identifier>ISSN: 0961-8368</identifier><identifier>EISSN: 1469-896X</identifier><identifier>DOI: 10.1110/ps.03455604</identifier><identifier>PMID: 15322283</identifier><language>eng</language><publisher>Bristol: Cold Spring Harbor Laboratory Press</publisher><subject>Amino Acid Sequence ; Animals ; CD55 Antigens - chemistry ; CD55 Antigens - genetics ; CD55 Antigens - metabolism ; CD55 Antigens - pharmacology ; Cell Membrane - metabolism ; Cells, Cultured ; CHAPS, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate ; complement ; Complement Activation - drug effects ; Complement C3a - antagonists & inhibitors ; Crystallization ; crystallography ; decay‐acceleration ; EcDAF, nonglycosylated human DAF 1–4 expressed in Escherichia coli ; EcDAF‐MP, soluble E. coli human DAF linked through a C‐terminal cysteine to the myristoylated peptide APT542 ; Escherichia coli - genetics ; GPI, glycophosphatidyl inositol ; Guinea Pigs ; Hemolysis - drug effects ; Humans ; Inclusion Bodies - genetics ; Inhibitory Concentration 50 ; membrane anchor ; Molecular Sequence Data ; nDAF, human native glycosylated (GPI‐anchored) DAF from erythrocytes ; PCR, polymerase chain reaction ; PpDAF, human DAF1–4 expressed in Pichia pastoris, N glycosylated and with an oligohistidine tag ; Protein Structure, Tertiary ; Rabbits ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; SCR, short consensus repeat ; TCEP, Tris‐(2‐carboxyethyl) phosphine ; X-Ray Diffraction</subject><ispartof>Protein science, 2004-09, Vol.13 (9), p.2406-2415</ispartof><rights>Copyright © 2004 The Protein Society</rights><rights>Copyright © Copyright 2004 The Protein Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3836-63ae3fb0d1c61d6c712f18541c0a02405ff496872ec8c6cf1584194c4eea767a3</citedby><cites>FETCH-LOGICAL-c3836-63ae3fb0d1c61d6c712f18541c0a02405ff496872ec8c6cf1584194c4eea767a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2280017/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2280017/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,1411,1427,27901,27902,45550,45551,46384,46808,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15322283$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>White, Jennifer</creatorcontrib><creatorcontrib>Lukacik, Petra</creatorcontrib><creatorcontrib>Esser, Dirk</creatorcontrib><creatorcontrib>Steward, Michael</creatorcontrib><creatorcontrib>Giddings, Naomi</creatorcontrib><creatorcontrib>Bright, Jeremy R.</creatorcontrib><creatorcontrib>Fritchley, Sarah J.</creatorcontrib><creatorcontrib>Morgan, B. Paul</creatorcontrib><creatorcontrib>Lea, Susan M.</creatorcontrib><creatorcontrib>Smith, Geoffrey P.</creatorcontrib><creatorcontrib>Smith, Richard A.G.</creatorcontrib><title>Biological activity, membrane‐targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli</title><title>Protein science</title><addtitle>Protein Sci</addtitle><description>Decay‐accelerating factor (DAF, CD55) is a glycophosphatidyl inositol‐anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane‐targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C‐terminal cysteine residue to permit site‐specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane‐localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF‐derived therapeutic. Crystals of the E. coli‐derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X‐ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>CD55 Antigens - chemistry</subject><subject>CD55 Antigens - genetics</subject><subject>CD55 Antigens - metabolism</subject><subject>CD55 Antigens - pharmacology</subject><subject>Cell Membrane - metabolism</subject><subject>Cells, Cultured</subject><subject>CHAPS, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate</subject><subject>complement</subject><subject>Complement Activation - drug effects</subject><subject>Complement C3a - antagonists & inhibitors</subject><subject>Crystallization</subject><subject>crystallography</subject><subject>decay‐acceleration</subject><subject>EcDAF, nonglycosylated human DAF 1–4 expressed in Escherichia coli</subject><subject>EcDAF‐MP, soluble E. coli human DAF linked through a C‐terminal cysteine to the myristoylated peptide APT542</subject><subject>Escherichia coli - genetics</subject><subject>GPI, glycophosphatidyl inositol</subject><subject>Guinea Pigs</subject><subject>Hemolysis - drug effects</subject><subject>Humans</subject><subject>Inclusion Bodies - genetics</subject><subject>Inhibitory Concentration 50</subject><subject>membrane anchor</subject><subject>Molecular Sequence Data</subject><subject>nDAF, human native glycosylated (GPI‐anchored) DAF from erythrocytes</subject><subject>PCR, polymerase chain reaction</subject><subject>PpDAF, human DAF1–4 expressed in Pichia pastoris, N glycosylated and with an oligohistidine tag</subject><subject>Protein Structure, Tertiary</subject><subject>Rabbits</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>SCR, short consensus repeat</subject><subject>TCEP, Tris‐(2‐carboxyethyl) phosphine</subject><subject>X-Ray Diffraction</subject><issn>0961-8368</issn><issn>1469-896X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kU1v1DAQhq0K1G4LJ-7Id5rFEztOckGiVVuQKrWqQOJmeZ3x1siJIztbCCfuvfQ39pdguuXrwsnS-JlnNPMS8gLYEgDY6zEtGRdVJZnYIQsQsi2aVn56QhaslVA0XDZ7ZD-lz4wxASXfJXtQ8bIsG74gt0cu-LB2RnuqzeRu3DQf0h77VdQD3n-_m3Rc4-SGNe1D52wGJxeGQ6qHjpo4p0l77749FGmwNAW_WXmk15teD7RDo-fsNegx6geLzVNCpPh1jJgSdtQN9GRJTfDuGXlqtU_4_PE9IB9PTz4cvyvOL87eH789LwzPyxSSa-R2xTowEjppaigtNJUAwzQrBausFa1s6hJNY6SxUDUCWmEEoq5lrfkBebP1jptVj53BYYraqzG6XsdZBe3Uvz-Du1brcKPyyRiDOgtebQUmhpQi2t-9wNTPTNSY1K9MMv3y73F_2McQMlBugS_O4_w_l7q8ugCed5T8B8Tfm88</recordid><startdate>200409</startdate><enddate>200409</enddate><creator>White, Jennifer</creator><creator>Lukacik, Petra</creator><creator>Esser, Dirk</creator><creator>Steward, Michael</creator><creator>Giddings, Naomi</creator><creator>Bright, Jeremy R.</creator><creator>Fritchley, Sarah J.</creator><creator>Morgan, B. Paul</creator><creator>Lea, Susan M.</creator><creator>Smith, Geoffrey P.</creator><creator>Smith, Richard A.G.</creator><general>Cold Spring Harbor Laboratory Press</general><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>5PM</scope></search><sort><creationdate>200409</creationdate><title>Biological activity, membrane‐targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli</title><author>White, Jennifer ; Lukacik, Petra ; Esser, Dirk ; Steward, Michael ; Giddings, Naomi ; Bright, Jeremy R. ; Fritchley, Sarah J. ; Morgan, B. Paul ; Lea, Susan M. ; Smith, Geoffrey P. ; Smith, Richard A.G.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3836-63ae3fb0d1c61d6c712f18541c0a02405ff496872ec8c6cf1584194c4eea767a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>CD55 Antigens - chemistry</topic><topic>CD55 Antigens - genetics</topic><topic>CD55 Antigens - metabolism</topic><topic>CD55 Antigens - pharmacology</topic><topic>Cell Membrane - metabolism</topic><topic>Cells, Cultured</topic><topic>CHAPS, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate</topic><topic>complement</topic><topic>Complement Activation - drug effects</topic><topic>Complement C3a - antagonists & inhibitors</topic><topic>Crystallization</topic><topic>crystallography</topic><topic>decay‐acceleration</topic><topic>EcDAF, nonglycosylated human DAF 1–4 expressed in Escherichia coli</topic><topic>EcDAF‐MP, soluble E. coli human DAF linked through a C‐terminal cysteine to the myristoylated peptide APT542</topic><topic>Escherichia coli - genetics</topic><topic>GPI, glycophosphatidyl inositol</topic><topic>Guinea Pigs</topic><topic>Hemolysis - drug effects</topic><topic>Humans</topic><topic>Inclusion Bodies - genetics</topic><topic>Inhibitory Concentration 50</topic><topic>membrane anchor</topic><topic>Molecular Sequence Data</topic><topic>nDAF, human native glycosylated (GPI‐anchored) DAF from erythrocytes</topic><topic>PCR, polymerase chain reaction</topic><topic>PpDAF, human DAF1–4 expressed in Pichia pastoris, N glycosylated and with an oligohistidine tag</topic><topic>Protein Structure, Tertiary</topic><topic>Rabbits</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>SCR, short consensus repeat</topic><topic>TCEP, Tris‐(2‐carboxyethyl) phosphine</topic><topic>X-Ray Diffraction</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>White, Jennifer</creatorcontrib><creatorcontrib>Lukacik, Petra</creatorcontrib><creatorcontrib>Esser, Dirk</creatorcontrib><creatorcontrib>Steward, Michael</creatorcontrib><creatorcontrib>Giddings, Naomi</creatorcontrib><creatorcontrib>Bright, Jeremy R.</creatorcontrib><creatorcontrib>Fritchley, Sarah J.</creatorcontrib><creatorcontrib>Morgan, B. Paul</creatorcontrib><creatorcontrib>Lea, Susan M.</creatorcontrib><creatorcontrib>Smith, Geoffrey P.</creatorcontrib><creatorcontrib>Smith, Richard A.G.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Protein science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>White, Jennifer</au><au>Lukacik, Petra</au><au>Esser, Dirk</au><au>Steward, Michael</au><au>Giddings, Naomi</au><au>Bright, Jeremy R.</au><au>Fritchley, Sarah J.</au><au>Morgan, B. Paul</au><au>Lea, Susan M.</au><au>Smith, Geoffrey P.</au><au>Smith, Richard A.G.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Biological activity, membrane‐targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli</atitle><jtitle>Protein science</jtitle><addtitle>Protein Sci</addtitle><date>2004-09</date><risdate>2004</risdate><volume>13</volume><issue>9</issue><spage>2406</spage><epage>2415</epage><pages>2406-2415</pages><issn>0961-8368</issn><eissn>1469-896X</eissn><abstract>Decay‐accelerating factor (DAF, CD55) is a glycophosphatidyl inositol‐anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane‐targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C‐terminal cysteine residue to permit site‐specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane‐localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF‐derived therapeutic. Crystals of the E. coli‐derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X‐ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.</abstract><cop>Bristol</cop><pub>Cold Spring Harbor Laboratory Press</pub><pmid>15322283</pmid><doi>10.1110/ps.03455604</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| source | Wiley Free Content; MEDLINE; Wiley Online Library Journals Frontfile Complete; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Amino Acid Sequence Animals CD55 Antigens - chemistry CD55 Antigens - genetics CD55 Antigens - metabolism CD55 Antigens - pharmacology Cell Membrane - metabolism Cells, Cultured CHAPS, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate complement Complement Activation - drug effects Complement C3a - antagonists & inhibitors Crystallization crystallography decay‐acceleration EcDAF, nonglycosylated human DAF 1–4 expressed in Escherichia coli EcDAF‐MP, soluble E. coli human DAF linked through a C‐terminal cysteine to the myristoylated peptide APT542 Escherichia coli - genetics GPI, glycophosphatidyl inositol Guinea Pigs Hemolysis - drug effects Humans Inclusion Bodies - genetics Inhibitory Concentration 50 membrane anchor Molecular Sequence Data nDAF, human native glycosylated (GPI‐anchored) DAF from erythrocytes PCR, polymerase chain reaction PpDAF, human DAF1–4 expressed in Pichia pastoris, N glycosylated and with an oligohistidine tag Protein Structure, Tertiary Rabbits Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism SCR, short consensus repeat TCEP, Tris‐(2‐carboxyethyl) phosphine X-Ray Diffraction |
| title | Biological activity, membrane‐targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli |
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