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
Hauptverfasser: Baranauskas, Aurimas, Paliksa, Sigitas, Alzbutas, Gediminas, Vaitkevicius, Mindaugas, Lubiene, Judita, Letukiene, Virginija, Burinskas, Sigitas, Sasnauskas, Giedrius, Skirgaila, Remigijus
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container_end_page 668
container_issue 10
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container_title Protein engineering, design and selection
container_volume 25
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. 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source Oxford University Press Journals All Titles (1996-Current); MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection
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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