RT Journal Article
SR Electronic
T1 Integrating Gene Synthesis and Microfluidic Protein Analysis for Rapid Protein Engineering
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 025239
DO 10.1101/025239
A1 Matthew C. Blackburn
A1 Ekaterina Petrova
A1 Bruno E. Correia
A1 Sebastian J. Maerkl
YR 2015
UL http://biorxiv.org/content/early/2015/08/21/025239.abstract
AB 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 fundamental studies in protein biophysics, and protein design/engineering.