Aug 21, 2015

Integrating Gene Synthesis and Microfluidic Protein Analysis for Rapid Protein Engineering

BioRxiv : the Preprint Server for Biology
Matthew C BlackburnSebastian J Maerkl

Abstract

The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology, and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification, and characterization (mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE- MITOMI we synthesized and characterized over 440 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF – DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate...Continue Reading

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Mentioned in this Paper

Study
Ape Diseases
Primer Extension
CREBZF protein, human
Synthetic Genes
Biophysics
Zinc
Oligonucleotide Primers
Binding (Molecular Function)
Purification Aspects

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