Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants
In vitro synthesis of cDNA is one of the most important techniques in present molecular biology. Faithful synthesis of long cDNA on highly structured RNA templates requires thermostable and processive reverse transcriptases. In a recent attempt to increase the thermostability of the wt Moloney Murin...
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Veröffentlicht in: | Protein engineering, design and selection design and selection, 2012-10, Vol.25 (10), p.657-668 |
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creator | Baranauskas, Aurimas Paliksa, Sigitas Alzbutas, Gediminas Vaitkevicius, Mindaugas Lubiene, Judita Letukiene, Virginija Burinskas, Sigitas Sasnauskas, Giedrius Skirgaila, Remigijus |
description | In vitro synthesis of cDNA is one of the most important techniques in present molecular biology. Faithful synthesis of long cDNA on highly structured RNA templates requires thermostable and processive reverse transcriptases. In a recent attempt to increase the thermostability of the wt Moloney Murine leukemia virus reverse transcriptase (M-MuLV RT), we have employed the compartmentalized ribosome display (CRD) evolution in vitro technique and identified a large set of previously unknown mutations that enabled cDNA synthesis at elevated temperatures. In this study, we have characterized a group of the M-MuLV RT variants (28 novel amino acid positions, 84 point mutants) carrying the individual mutations. The performance of point mutants (thermal inactivation rate, substrate-binding affinity and processivity) correlated remarkably well with the mutation selection frequency in the CRD experiment. By combining the best-performing mutations D200N, L603W, T330P, L139P and E607K, we have generated highly processive and thermostable multiply-mutated M-MuLV RT variants. The processivity of the best-performing multiple mutant increased to 1500 nt (65-fold improvement in comparison to the wt enzyme), and the maximum temperature of the full-length 7.5-kb cDNA synthesis was raised to 62°C (17° higher in comparison with the wt enzyme). |
doi_str_mv | 10.1093/protein/gzs034 |
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Faithful synthesis of long cDNA on highly structured RNA templates requires thermostable and processive reverse transcriptases. In a recent attempt to increase the thermostability of the wt Moloney Murine leukemia virus reverse transcriptase (M-MuLV RT), we have employed the compartmentalized ribosome display (CRD) evolution in vitro technique and identified a large set of previously unknown mutations that enabled cDNA synthesis at elevated temperatures. In this study, we have characterized a group of the M-MuLV RT variants (28 novel amino acid positions, 84 point mutants) carrying the individual mutations. The performance of point mutants (thermal inactivation rate, substrate-binding affinity and processivity) correlated remarkably well with the mutation selection frequency in the CRD experiment. By combining the best-performing mutations D200N, L603W, T330P, L139P and E607K, we have generated highly processive and thermostable multiply-mutated M-MuLV RT variants. The processivity of the best-performing multiple mutant increased to 1500 nt (65-fold improvement in comparison to the wt enzyme), and the maximum temperature of the full-length 7.5-kb cDNA synthesis was raised to 62°C (17° higher in comparison with the wt enzyme).</description><identifier>ISSN: 1741-0126</identifier><identifier>EISSN: 1741-0134</identifier><identifier>DOI: 10.1093/protein/gzs034</identifier><identifier>PMID: 22691702</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Animals ; DNA, Complementary - genetics ; Mice ; Moloney murine leukemia virus ; Moloney murine leukemia virus - enzymology ; Moloney murine leukemia virus - genetics ; Moloney murine leukemia virus - metabolism ; Mutagenesis ; Point Mutation ; Protein Stability ; RNA-Directed DNA Polymerase - chemistry ; RNA-Directed DNA Polymerase - genetics ; RNA-Directed DNA Polymerase - metabolism ; Temperature</subject><ispartof>Protein engineering, design and selection, 2012-10, Vol.25 (10), p.657-668</ispartof><rights>The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c468t-8c7386ccfbd056a7d41058df433cff79331b25055bf615827397aca1deec6acf3</citedby><cites>FETCH-LOGICAL-c468t-8c7386ccfbd056a7d41058df433cff79331b25055bf615827397aca1deec6acf3</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/22691702$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Baranauskas, Aurimas</creatorcontrib><creatorcontrib>Paliksa, Sigitas</creatorcontrib><creatorcontrib>Alzbutas, Gediminas</creatorcontrib><creatorcontrib>Vaitkevicius, Mindaugas</creatorcontrib><creatorcontrib>Lubiene, Judita</creatorcontrib><creatorcontrib>Letukiene, Virginija</creatorcontrib><creatorcontrib>Burinskas, Sigitas</creatorcontrib><creatorcontrib>Sasnauskas, Giedrius</creatorcontrib><creatorcontrib>Skirgaila, Remigijus</creatorcontrib><title>Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants</title><title>Protein engineering, design and selection</title><addtitle>Protein Eng Des Sel</addtitle><description>In vitro synthesis of cDNA is one of the most important techniques in present molecular biology. Faithful synthesis of long cDNA on highly structured RNA templates requires thermostable and processive reverse transcriptases. In a recent attempt to increase the thermostability of the wt Moloney Murine leukemia virus reverse transcriptase (M-MuLV RT), we have employed the compartmentalized ribosome display (CRD) evolution in vitro technique and identified a large set of previously unknown mutations that enabled cDNA synthesis at elevated temperatures. In this study, we have characterized a group of the M-MuLV RT variants (28 novel amino acid positions, 84 point mutants) carrying the individual mutations. The performance of point mutants (thermal inactivation rate, substrate-binding affinity and processivity) correlated remarkably well with the mutation selection frequency in the CRD experiment. By combining the best-performing mutations D200N, L603W, T330P, L139P and E607K, we have generated highly processive and thermostable multiply-mutated M-MuLV RT variants. The processivity of the best-performing multiple mutant increased to 1500 nt (65-fold improvement in comparison to the wt enzyme), and the maximum temperature of the full-length 7.5-kb cDNA synthesis was raised to 62°C (17° higher in comparison with the wt enzyme).</description><subject>Animals</subject><subject>DNA, Complementary - genetics</subject><subject>Mice</subject><subject>Moloney murine leukemia virus</subject><subject>Moloney murine leukemia virus - enzymology</subject><subject>Moloney murine leukemia virus - genetics</subject><subject>Moloney murine leukemia virus - metabolism</subject><subject>Mutagenesis</subject><subject>Point Mutation</subject><subject>Protein Stability</subject><subject>RNA-Directed DNA Polymerase - chemistry</subject><subject>RNA-Directed DNA Polymerase - genetics</subject><subject>RNA-Directed DNA Polymerase - metabolism</subject><subject>Temperature</subject><issn>1741-0126</issn><issn>1741-0134</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkT1PwzAQhi0EoqWwMqKMMKT1V-x0RAgKUisWYI0c59wYpUmxnaL21xNI6drpPvTce6d7EbomeEzwlE3Wrglg68ly5zHjJ2hIJCcxJoyfHnIqBujC-0-MqZCEnKMBpWJKJKZD1MygBqeCbepI1UWkS-WUDuDsrm82JqrhOyrtsqy2USjBrRofVF7BH9_t1-C93UC0iBft_CNysAHnIQpO1V47uw6qqzbKWVUHf4nOjKo8XO3jCL0_Pb49PMfz19nLw_081lykIU61ZKnQ2uQFToSSBSc4SQvDGdPGyCljJKcJTpLcCJKkVLKpVFqRAkALpQ0bodtetzvwqwUfspX1GqpK1dC0PiOccUwl5elxFKdYsO5btEPHPapd470Dk62dXSm37aDs149s70fW-9EN3Oy123wFxQH_N6AD7nqgadfHxH4AZVWZkA</recordid><startdate>20121001</startdate><enddate>20121001</enddate><creator>Baranauskas, Aurimas</creator><creator>Paliksa, Sigitas</creator><creator>Alzbutas, Gediminas</creator><creator>Vaitkevicius, Mindaugas</creator><creator>Lubiene, Judita</creator><creator>Letukiene, Virginija</creator><creator>Burinskas, Sigitas</creator><creator>Sasnauskas, Giedrius</creator><creator>Skirgaila, Remigijus</creator><general>Oxford University 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>7X8</scope><scope>7QO</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>20121001</creationdate><title>Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants</title><author>Baranauskas, Aurimas ; Paliksa, Sigitas ; Alzbutas, Gediminas ; Vaitkevicius, Mindaugas ; Lubiene, Judita ; Letukiene, Virginija ; Burinskas, Sigitas ; Sasnauskas, Giedrius ; Skirgaila, Remigijus</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c468t-8c7386ccfbd056a7d41058df433cff79331b25055bf615827397aca1deec6acf3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Animals</topic><topic>DNA, Complementary - genetics</topic><topic>Mice</topic><topic>Moloney murine leukemia virus</topic><topic>Moloney murine leukemia virus - enzymology</topic><topic>Moloney murine leukemia virus - genetics</topic><topic>Moloney murine leukemia virus - metabolism</topic><topic>Mutagenesis</topic><topic>Point Mutation</topic><topic>Protein Stability</topic><topic>RNA-Directed DNA Polymerase - chemistry</topic><topic>RNA-Directed DNA Polymerase - genetics</topic><topic>RNA-Directed DNA Polymerase - metabolism</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Baranauskas, Aurimas</creatorcontrib><creatorcontrib>Paliksa, Sigitas</creatorcontrib><creatorcontrib>Alzbutas, Gediminas</creatorcontrib><creatorcontrib>Vaitkevicius, Mindaugas</creatorcontrib><creatorcontrib>Lubiene, Judita</creatorcontrib><creatorcontrib>Letukiene, Virginija</creatorcontrib><creatorcontrib>Burinskas, Sigitas</creatorcontrib><creatorcontrib>Sasnauskas, Giedrius</creatorcontrib><creatorcontrib>Skirgaila, Remigijus</creatorcontrib><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><collection>Biotechnology Research Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Protein engineering, design and selection</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Baranauskas, Aurimas</au><au>Paliksa, Sigitas</au><au>Alzbutas, Gediminas</au><au>Vaitkevicius, Mindaugas</au><au>Lubiene, Judita</au><au>Letukiene, Virginija</au><au>Burinskas, Sigitas</au><au>Sasnauskas, Giedrius</au><au>Skirgaila, Remigijus</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants</atitle><jtitle>Protein engineering, design and selection</jtitle><addtitle>Protein Eng Des Sel</addtitle><date>2012-10-01</date><risdate>2012</risdate><volume>25</volume><issue>10</issue><spage>657</spage><epage>668</epage><pages>657-668</pages><issn>1741-0126</issn><eissn>1741-0134</eissn><abstract>In vitro synthesis of cDNA is one of the most important techniques in present molecular biology. Faithful synthesis of long cDNA on highly structured RNA templates requires thermostable and processive reverse transcriptases. In a recent attempt to increase the thermostability of the wt Moloney Murine leukemia virus reverse transcriptase (M-MuLV RT), we have employed the compartmentalized ribosome display (CRD) evolution in vitro technique and identified a large set of previously unknown mutations that enabled cDNA synthesis at elevated temperatures. In this study, we have characterized a group of the M-MuLV RT variants (28 novel amino acid positions, 84 point mutants) carrying the individual mutations. The performance of point mutants (thermal inactivation rate, substrate-binding affinity and processivity) correlated remarkably well with the mutation selection frequency in the CRD experiment. By combining the best-performing mutations D200N, L603W, T330P, L139P and E607K, we have generated highly processive and thermostable multiply-mutated M-MuLV RT variants. 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subjects | Animals DNA, Complementary - genetics Mice Moloney murine leukemia virus Moloney murine leukemia virus - enzymology Moloney murine leukemia virus - genetics Moloney murine leukemia virus - metabolism Mutagenesis Point Mutation Protein Stability RNA-Directed DNA Polymerase - chemistry RNA-Directed DNA Polymerase - genetics RNA-Directed DNA Polymerase - metabolism Temperature |
title | Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants |
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