Transcription factors control gene expression in all life. This raises the question of what is the smallest protein that can support such activity. Recent advances in protein directed evolution, using continuous phage evolution allow us to probe such questions in a new way. We have developed a method of Phagemid-Assisted Continuous Evolution (PACEmid) where the link between a mutant protein's activity and phage replication in vivo, allows evolution to occur. The main advantages of using phagemids are to facilitate library screening and to prevent the evolution of "cheaters" by constantly replenishing phage helper plasmids and conditional selection plasmids. This allows us to evolve new functions such as minimal dual transcription factors in E. coli. In nature, Cro from bacteriophage λ is one of the smallest known repressors (66 amino acids; a.a.), whereas activators are typically much larger (e.g. λ cI, 237 a.a.). Indeed, previous efforts to engineer a minimal activator from λ Cro resulted in no activity in vivo, in cells. In this presentation, we show how PACEmid directed evolution results in a new Cro dual activator-repressor that functions as efficiently as λ cI, in vivo. We find that a peptide as small as 63 a.a. functions efficiently as a transcription factor. To our knowledge, this is the smallest protein activator that enables polymerase recruitment, highlighting the capacity of transcription factors to evolve from very short peptide sequences.
Mark Isalan carried out a Ph.D. in engineering zinc fingers to bind new DNA sequences at the MRC LMB, in the University of Cambridge UK,1996-2000. This work was supervised by Prof. Sir Aaron Klug, OM, FRS, and continued postdoctorally from 2000-2002 at Gendaq Ltd, UK (now owned by Sangamo Biosciences, Richmond CA). The work ultimately contributed to the CompoZr zinc finger nucleases now available commercially from Sigma Aldrich. From 2002-2006 Dr. Isalan was awarded a Wellcome Trust International Research Fellowship to carry out research on engineering artificial gene networks in Prof. Luis Serrano's group at the EMBL Heidelberg, Germany. From 2006-2013 he was a group leader at the EMBL-CRG Systems Biology Unit in Barcelona, specialising in synthetic gene network engineering. He moved to Imperial College London in 2013 and continues to work in protein and gene network engineering, aiming to design biological systems that behave predictably and robustly.
For details of his research and recent publication, please visit HERE
© Copyright International BioDesign Research Conference - All rights reserved
