Teaching TetR to recognize a new inducer

Publikation: Beiträge in ZeitschriftenZeitschriftenaufsätzeForschungbegutachtet

Standard

Teaching TetR to recognize a new inducer. / Scholz, Oliver; Köstner, Martin; Reich, Marco et al.
in: Journal of Molecular Biology, Jahrgang 329, Nr. 2, 30.05.2003, S. 217-227.

Publikation: Beiträge in ZeitschriftenZeitschriftenaufsätzeForschungbegutachtet

Harvard

Scholz, O, Köstner, M, Reich, M, Gastiger, S & Hillen, W 2003, 'Teaching TetR to recognize a new inducer', Journal of Molecular Biology, Jg. 329, Nr. 2, S. 217-227. https://doi.org/10.1016/S0022-2836(03)00427-3

APA

Vancouver

Scholz O, Köstner M, Reich M, Gastiger S, Hillen W. Teaching TetR to recognize a new inducer. Journal of Molecular Biology. 2003 Mai 30;329(2):217-227. doi: 10.1016/S0022-2836(03)00427-3

Bibtex

@article{92f54383cef5442c87430a0618db279a,
title = "Teaching TetR to recognize a new inducer",
abstract = "Tet Repressor (TetR) recognizes the inducer tetracycline (tc) with high affinity. The tc analog 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) is not an inducer for TetR. Induction specificity for cmt3 was generated by employing a directed evolution approach to screen appropriate TetR mutants in four successive steps. The specificity of the best TetR mutant is more than 20,000-fold increased for cmt3 over tc as judged by the ratio of their respective binding constants. Two rounds of directed evolution via DNA shuffling revealed His64 as a key residue for inducer specificity. The best TetR mutant with cmt3 specificity contains the H64K exchange, leading to a 300-fold decreased tc and a 20-fold increased cmt3 affinity. Another round of directed evolution made use of randomized oligonucleotides to mutate selected residues close to the tc-binding pocket of TetR and yielded TetR S135L with a 250-fold increased cmt3 affinity. The double mutant TetR H64K S135L was constructed and again subjected to directed evolution using randomized oligonucleotides to alter residues in the {"}secondary shell{"} of the tc-binding pocket. The resulting best mutants TetR H64K E114Q S135L, TetR A61V H64K Q109E Q116E S135L and TetR H64K T112K S135L are fully inducible by cmt3 and not by tc. Thus, their inducer specificity has been redesigned. The molecular mechanism of changed inducer recognition is discussed, based on binding constants with several tc analogs and in light of the TetR crystal structure.",
keywords = "cmt3, Inducer specificity, Induction, Tet repressor, Tetracycline, Chemistry",
author = "Oliver Scholz and Martin K{\"o}stner and Marco Reich and Susanne Gastiger and Wolfgang Hillen",
note = "We thank Brad Zerler, CollaGenex, PA, for providing cmt1 and cmt3, and Mark Nelson, Paratek, Boston, MA, for san. This work was supported by the Deutsche Forschungsgemeinschaft through the SFB 473 and the Fonds der Chemischen Industrie. O.S. obtained a personal grant from the the Fonds der Chemischen Industrie.",
year = "2003",
month = may,
day = "30",
doi = "10.1016/S0022-2836(03)00427-3",
language = "English",
volume = "329",
pages = "217--227",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "2",

}

RIS

TY - JOUR

T1 - Teaching TetR to recognize a new inducer

AU - Scholz, Oliver

AU - Köstner, Martin

AU - Reich, Marco

AU - Gastiger, Susanne

AU - Hillen, Wolfgang

N1 - We thank Brad Zerler, CollaGenex, PA, for providing cmt1 and cmt3, and Mark Nelson, Paratek, Boston, MA, for san. This work was supported by the Deutsche Forschungsgemeinschaft through the SFB 473 and the Fonds der Chemischen Industrie. O.S. obtained a personal grant from the the Fonds der Chemischen Industrie.

PY - 2003/5/30

Y1 - 2003/5/30

N2 - Tet Repressor (TetR) recognizes the inducer tetracycline (tc) with high affinity. The tc analog 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) is not an inducer for TetR. Induction specificity for cmt3 was generated by employing a directed evolution approach to screen appropriate TetR mutants in four successive steps. The specificity of the best TetR mutant is more than 20,000-fold increased for cmt3 over tc as judged by the ratio of their respective binding constants. Two rounds of directed evolution via DNA shuffling revealed His64 as a key residue for inducer specificity. The best TetR mutant with cmt3 specificity contains the H64K exchange, leading to a 300-fold decreased tc and a 20-fold increased cmt3 affinity. Another round of directed evolution made use of randomized oligonucleotides to mutate selected residues close to the tc-binding pocket of TetR and yielded TetR S135L with a 250-fold increased cmt3 affinity. The double mutant TetR H64K S135L was constructed and again subjected to directed evolution using randomized oligonucleotides to alter residues in the "secondary shell" of the tc-binding pocket. The resulting best mutants TetR H64K E114Q S135L, TetR A61V H64K Q109E Q116E S135L and TetR H64K T112K S135L are fully inducible by cmt3 and not by tc. Thus, their inducer specificity has been redesigned. The molecular mechanism of changed inducer recognition is discussed, based on binding constants with several tc analogs and in light of the TetR crystal structure.

AB - Tet Repressor (TetR) recognizes the inducer tetracycline (tc) with high affinity. The tc analog 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) is not an inducer for TetR. Induction specificity for cmt3 was generated by employing a directed evolution approach to screen appropriate TetR mutants in four successive steps. The specificity of the best TetR mutant is more than 20,000-fold increased for cmt3 over tc as judged by the ratio of their respective binding constants. Two rounds of directed evolution via DNA shuffling revealed His64 as a key residue for inducer specificity. The best TetR mutant with cmt3 specificity contains the H64K exchange, leading to a 300-fold decreased tc and a 20-fold increased cmt3 affinity. Another round of directed evolution made use of randomized oligonucleotides to mutate selected residues close to the tc-binding pocket of TetR and yielded TetR S135L with a 250-fold increased cmt3 affinity. The double mutant TetR H64K S135L was constructed and again subjected to directed evolution using randomized oligonucleotides to alter residues in the "secondary shell" of the tc-binding pocket. The resulting best mutants TetR H64K E114Q S135L, TetR A61V H64K Q109E Q116E S135L and TetR H64K T112K S135L are fully inducible by cmt3 and not by tc. Thus, their inducer specificity has been redesigned. The molecular mechanism of changed inducer recognition is discussed, based on binding constants with several tc analogs and in light of the TetR crystal structure.

KW - cmt3

KW - Inducer specificity

KW - Induction

KW - Tet repressor

KW - Tetracycline

KW - Chemistry

UR - http://www.scopus.com/inward/record.url?scp=0038737990&partnerID=8YFLogxK

U2 - 10.1016/S0022-2836(03)00427-3

DO - 10.1016/S0022-2836(03)00427-3

M3 - Journal articles

C2 - 12758071

AN - SCOPUS:0038737990

VL - 329

SP - 217

EP - 227

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -

DOI