Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA Polymerase

Holzberger, Bastian; Marx, Andreas

Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA Polymerase

Dateien

Holzberger_JACS_2010.pdfGröße: 1.61 MBDownloads: 488

Datum

2010

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Chemie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

Journal of the American Chemical Society : JACS. 2010, 132(44), pp. 15708-15713. ISSN 0002-7863. eISSN 1520-5126. Available under: doi: 10.1021/ja106525y

Zusammenfassung

Protein engineering may be achieved by rational design, directed evolution-based methods, or computational protein design. Mostly these methods make recourse to the restricted pool of the 20 natural amino acids. With the ability to introduce different new kinds of functionalities into proteins, the use of noncanonical amino acids became a promising new method in protein engineering. Here, we report on the generation of a multifluorinated DNA polymerase. DNA polymerases are highly dynamic enzymes that catalyze DNA synthesis in a template-dependent manner, thereby passing several conformational states during the catalytic cycle. Here, we globally replaced 32 proline residues by the noncanonical imino acid (4R)-fluoroproline in a DNA polymerase of 540 amino acids (KlenTaq DNA polymerase). Interestingly, the substitution level of the proline residues was very efficient (92%). Nonetheless, the introduction of (4R)-fluoroproline into the DNA polymerase resulted in a highly active fluorinated enzyme, which was investigated in primer extension and PCR assays to analyze activity, selectivity, and stability in comparison to the parental enzyme. The DNA polymerase retained fidelity, activity, and sensitivity as the parental wild-type enzyme accompanied by some loss in thermostability. These results demonstrate that a noncanonical amino acid can be used for substitutions of natural counterparts in a highly dynamic enzyme with high molecular weight without effecting crucial enzyme properties. Furthermore, the employed DNA polymerase represents a promising starting point for directed DNA polymerase evolution with noncanonical amino acids.

Fachgebiet (DDC)

540 Chemie

Schlagwörter

Protein engineering, DNA polymerases

Zitieren

ISO 690

HOLZBERGER, Bastian, Andreas MARX, 2010. Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA Polymerase. In: Journal of the American Chemical Society : JACS. 2010, 132(44), pp. 15708-15713. ISSN 0002-7863. eISSN 1520-5126. Available under: doi: 10.1021/ja106525y

BibTex

@article{Holzberger2010Repla-9818,
  year={2010},
  doi={10.1021/ja106525y},
  title={Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA Polymerase},
  number={44},
  volume={132},
  issn={0002-7863},
  journal={Journal of the American Chemical Society : JACS},
  pages={15708--15713},
  author={Holzberger, Bastian and Marx, Andreas}
}

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Universitätsbibliographie

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