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A high-throughput microfluidic method for generating and characterizing transcription factor mutant libraries


Geertz, Marcel; Rockel, Sylvie; Maerkl, Sebastian J (2012). A high-throughput microfluidic method for generating and characterizing transcription factor mutant libraries. Methods in Molecular Biology, 813:107-123.

Abstract

Characterizing libraries of mutant proteins is a challenging task, but can lead to detailed functional insights on a specific protein, and general insights for families of proteins such as transcription factors. Challenges in mutant protein screening consist in synthesizing the necessary expression ready DNA constructs and transforming them into a suitable host for protein expression. Protein purification and characterization are also non-trivial tasks that are not easily scalable to hundreds or thousands of protein variants. Here we describe a method based on a high-throughput microfluidic platform to screen and characterize the binding profile of hundreds of transcription factor variants. DNA constructs are synthesized by a rapid two-step PCR approach without the need of cloning or transformation steps. All transcription factor mutants are expressed on-chip followed by characterization of their binding specificities against 64 different DNA target sequences. The current microfluidic platformcan synthesize and characterize up to 2400 protein–DNA pairs in parallel. The platform method is also generally applicable, allowing high-throughput functional studies of proteins.

Characterizing libraries of mutant proteins is a challenging task, but can lead to detailed functional insights on a specific protein, and general insights for families of proteins such as transcription factors. Challenges in mutant protein screening consist in synthesizing the necessary expression ready DNA constructs and transforming them into a suitable host for protein expression. Protein purification and characterization are also non-trivial tasks that are not easily scalable to hundreds or thousands of protein variants. Here we describe a method based on a high-throughput microfluidic platform to screen and characterize the binding profile of hundreds of transcription factor variants. DNA constructs are synthesized by a rapid two-step PCR approach without the need of cloning or transformation steps. All transcription factor mutants are expressed on-chip followed by characterization of their binding specificities against 64 different DNA target sequences. The current microfluidic platformcan synthesize and characterize up to 2400 protein–DNA pairs in parallel. The platform method is also generally applicable, allowing high-throughput functional studies of proteins.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:Special Collections > SystemsX.ch
Special Collections > SystemsX.ch > Research, Technology and Development Projects > DynamiX
Special Collections > SystemsX.ch > Research, Technology and Development Projects
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:January 2012
Deposited On:22 Jun 2013 14:42
Last Modified:05 Apr 2016 16:49
Publisher:Humana Press
ISSN:1064-3745
Publisher DOI:https://doi.org/10.1007/978-1-61779-412-4_6
Permanent URL: https://doi.org/10.5167/uzh-78707

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