Volume 6, Issue 6 p. 1062-1067
Full Paper

Multiplex-PCR-Based Recombination as a Novel High-Fidelity Method for Directed Evolution

Thorsten Eggert Dr.

Thorsten Eggert Dr.

Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, 52426 Jülich, Germany, Fax: (+49) 2461-61-2490

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Susanne Aileen Funke Dr.

Susanne Aileen Funke Dr.

Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, 52426 Jülich, Germany, Fax: (+49) 2461-61-2490

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Nalam M. Rao Dr.

Nalam M. Rao Dr.

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

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Priyamvada Acharya Dr.

Priyamvada Acharya Dr.

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

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Holger Krumm Dr.

Holger Krumm Dr.

Max-Planck Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany

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Manfred T. Reetz Prof. Dr.

Manfred T. Reetz Prof. Dr.

Max-Planck Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany

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Karl-Erich Jaeger Prof. Dr.

Karl-Erich Jaeger Prof. Dr.

Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, 52426 Jülich, Germany, Fax: (+49) 2461-61-2490

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First published: 30 May 2005
Citations: 24

Graphical Abstract

Directed evolution has matured during the last decade to become a key technology in the field of molecular enzyme engineering. We present a novel in vitro recombination method called multiplex-PCR-based recombination (MUPREC) which can be used to recombine single point mutations previously generated by error-prone PCR or complete saturation mutagenesis (see graphic).

Abstract

A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex-PCR) generates gene fragments that contain preformed point mutations. These fragments are subsequently assembled into full-length genes by a recombination-PCR step. The process of multiplex-PCR-based recombination (MUPREC) does not require DNase I digestion for gene-fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild-type background. Furthermore, the low error rate results in high-quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso-compound. Thereby, the method was proved to be useful in producing a reliable second-generation library of true recombinants from which better performing variants were identified by using a high-throughput electrospray ionization mass spectrometry (ESI-MS) screening system.