Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes
The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein–protein adaptors; members of the LMO family each cont...
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Veröffentlicht in: | Protein engineering 2001-07, Vol.14 (7), p.493-499 |
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description | The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein–protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO–ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional 1H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO–ldb1 interactions. |
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LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein–protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO–ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional 1H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO–ldb1 interactions.</description><identifier>ISSN: 0269-2139</identifier><identifier>ISSN: 1741-0126</identifier><identifier>EISSN: 1460-213X</identifier><identifier>EISSN: 1741-0134</identifier><identifier>DOI: 10.1093/protein/14.7.493</identifier><identifier>PMID: 11522923</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Adaptor Proteins, Signal Transducing ; Amino Acid Sequence ; Animals ; Binding Sites ; Cloning, Molecular ; DNA-Binding Proteins - chemistry ; DNA-Binding Proteins - isolation & purification ; Escherichia coli - genetics ; fusion protein ; Homeodomain Proteins - chemistry ; Homeodomain Proteins - isolation & purification ; ldb1 ; LIM Domain Proteins ; LMO transcription factors ; Magnetic Resonance Spectroscopy ; Metalloproteins - chemistry ; Metalloproteins - isolation & purification ; Mice ; Nuclear Proteins - chemistry ; Protein Binding ; Protein Engineering ; Protein Folding ; Protein Structure, Secondary ; Recombinant Fusion Proteins ; Transcription Factors - chemistry ; Transcription Factors - isolation & purification ; Zinc - chemistry</subject><ispartof>Protein engineering, 2001-07, Vol.14 (7), p.493-499</ispartof><rights>Oxford University Press 2001</rights><rights>Copyright Oxford University Press(England) Jul 2001</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c469t-f5ed09c18d7e79fad70d74a982ecf0009462666c0ed8b3b7bb5c5d3bae0f06cc3</citedby><cites>FETCH-LOGICAL-c469t-f5ed09c18d7e79fad70d74a982ecf0009462666c0ed8b3b7bb5c5d3bae0f06cc3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,1578,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11522923$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Deane, Janet E.</creatorcontrib><creatorcontrib>Sum, Eleanor</creatorcontrib><creatorcontrib>Mackay, Joel P.</creatorcontrib><creatorcontrib>Lindeman, Geoffrey J.</creatorcontrib><creatorcontrib>Visvader, Jane E.</creatorcontrib><creatorcontrib>Matthews, Jacqueline M.</creatorcontrib><title>Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes</title><title>Protein engineering</title><addtitle>Protein Eng</addtitle><addtitle>Protein Eng</addtitle><description>The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein–protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO–ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional 1H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO–ldb1 interactions.</description><subject>Adaptor Proteins, Signal Transducing</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Binding Sites</subject><subject>Cloning, Molecular</subject><subject>DNA-Binding Proteins - chemistry</subject><subject>DNA-Binding Proteins - isolation & purification</subject><subject>Escherichia coli - genetics</subject><subject>fusion protein</subject><subject>Homeodomain Proteins - chemistry</subject><subject>Homeodomain Proteins - isolation & purification</subject><subject>ldb1</subject><subject>LIM Domain Proteins</subject><subject>LMO transcription factors</subject><subject>Magnetic Resonance Spectroscopy</subject><subject>Metalloproteins - chemistry</subject><subject>Metalloproteins - isolation & purification</subject><subject>Mice</subject><subject>Nuclear Proteins - chemistry</subject><subject>Protein Binding</subject><subject>Protein Engineering</subject><subject>Protein Folding</subject><subject>Protein Structure, Secondary</subject><subject>Recombinant Fusion Proteins</subject><subject>Transcription Factors - chemistry</subject><subject>Transcription Factors - isolation & purification</subject><subject>Zinc - chemistry</subject><issn>0269-2139</issn><issn>1741-0126</issn><issn>1460-213X</issn><issn>1741-0134</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc1rFDEYh4Modq3ePcngwYvONl-TbHqTrf2A1YIoLF5CJnlnO3UmmSYzUP3rzXQXBS96ykvyvA_88kPoJcFLghU7GWIYofUnhC_lkiv2CC0IF7ikhG0fowWmQs2zOkLPUrrFGK-wok_RESEVpYqyBRrOILU7_67IKjfZsQ2-MN4V9sZEY0eI7U_zcBma4nxz9Yk-vM4TPy3GGyjA71oPmfO7mWn9GE0fOrBTZ2LRuZqcbj5eFzb0Qwf3kJ6jJ43pErw4nMfo6_mHL-vLcnN9cbV-vyktF2osmwocVpasnASpGuMkdpIbtaJgmxxEcUGFEBaDW9WslnVd2cqx2gBusLCWHaM3e28OdjdBGnXfJgtdZzyEKWlJCBWMqX-CFHPBKz6Dr_8Cb8MUfQ6hKa14_lrCMoT3kI0hpQiNHmLbm_hDE6znzvShM024ljpr88qrg3eqe3B_Fg4lZeDtHgjT8D-6ck-3aYT737yJ37WQTFb6cvtNr7dn5KISnzVjvwBhe7GQ</recordid><startdate>20010701</startdate><enddate>20010701</enddate><creator>Deane, Janet E.</creator><creator>Sum, Eleanor</creator><creator>Mackay, Joel P.</creator><creator>Lindeman, Geoffrey J.</creator><creator>Visvader, Jane E.</creator><creator>Matthews, Jacqueline M.</creator><general>Oxford University Press</general><general>Oxford Publishing Limited (England)</general><scope>BSCLL</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>7QL</scope><scope>7QO</scope><scope>7QP</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20010701</creationdate><title>Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes</title><author>Deane, Janet E. ; Sum, Eleanor ; Mackay, Joel P. ; Lindeman, Geoffrey J. ; Visvader, Jane E. ; Matthews, Jacqueline M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c469t-f5ed09c18d7e79fad70d74a982ecf0009462666c0ed8b3b7bb5c5d3bae0f06cc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Adaptor Proteins, Signal Transducing</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Binding Sites</topic><topic>Cloning, Molecular</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>DNA-Binding Proteins - isolation & purification</topic><topic>Escherichia coli - genetics</topic><topic>fusion protein</topic><topic>Homeodomain Proteins - chemistry</topic><topic>Homeodomain Proteins - isolation & purification</topic><topic>ldb1</topic><topic>LIM Domain Proteins</topic><topic>LMO transcription factors</topic><topic>Magnetic Resonance Spectroscopy</topic><topic>Metalloproteins - chemistry</topic><topic>Metalloproteins - isolation & purification</topic><topic>Mice</topic><topic>Nuclear Proteins - chemistry</topic><topic>Protein Binding</topic><topic>Protein Engineering</topic><topic>Protein Folding</topic><topic>Protein Structure, Secondary</topic><topic>Recombinant Fusion Proteins</topic><topic>Transcription Factors - chemistry</topic><topic>Transcription Factors - isolation & purification</topic><topic>Zinc - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Deane, Janet E.</creatorcontrib><creatorcontrib>Sum, Eleanor</creatorcontrib><creatorcontrib>Mackay, Joel P.</creatorcontrib><creatorcontrib>Lindeman, Geoffrey J.</creatorcontrib><creatorcontrib>Visvader, Jane E.</creatorcontrib><creatorcontrib>Matthews, Jacqueline M.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Protein engineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Deane, Janet E.</au><au>Sum, Eleanor</au><au>Mackay, Joel P.</au><au>Lindeman, Geoffrey J.</au><au>Visvader, Jane E.</au><au>Matthews, Jacqueline M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes</atitle><jtitle>Protein engineering</jtitle><stitle>Protein Eng</stitle><addtitle>Protein Eng</addtitle><date>2001-07-01</date><risdate>2001</risdate><volume>14</volume><issue>7</issue><spage>493</spage><epage>499</epage><pages>493-499</pages><issn>0269-2139</issn><issn>1741-0126</issn><eissn>1460-213X</eissn><eissn>1741-0134</eissn><abstract>The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein–protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO–ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional 1H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO–ldb1 interactions.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>11522923</pmid><doi>10.1093/protein/14.7.493</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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source | Oxford University Press Journals All Titles (1996-Current); MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals |
subjects | Adaptor Proteins, Signal Transducing Amino Acid Sequence Animals Binding Sites Cloning, Molecular DNA-Binding Proteins - chemistry DNA-Binding Proteins - isolation & purification Escherichia coli - genetics fusion protein Homeodomain Proteins - chemistry Homeodomain Proteins - isolation & purification ldb1 LIM Domain Proteins LMO transcription factors Magnetic Resonance Spectroscopy Metalloproteins - chemistry Metalloproteins - isolation & purification Mice Nuclear Proteins - chemistry Protein Binding Protein Engineering Protein Folding Protein Structure, Secondary Recombinant Fusion Proteins Transcription Factors - chemistry Transcription Factors - isolation & purification Zinc - chemistry |
title | Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes |
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