Download - Michael A. Fisher, Kara L. McKinley, Luke H. Bradley, Sara R. Viola, and Michael H. Hecht
De Novo Designed Proteins from a Library of Artificial Sequences Function
in Escherichia Coli and Enable Cell Growth
Michael A. Fisher, Kara L. McKinley, Luke H. Bradley, Sara R. Viola, and Michael H. Hecht
PLoS ONE: January 4, 2011
Presented by: Amber Lin & Yuan Zhao
"Must the toolkit of life be so restricted?"
• 20100 possible 100-residue proteinso life sustained by only 4,000 in E.coli, ~20,000 in humans
• challenge of synthetic biology: develop living parts independent of nature
• generated novel 102-residue 4-helix bundles using binary pattern
Binary Pattern• Strategy that this specific pattern will coincide with structure
of natural, folded proteins
• Pattern of PNPPNNPPNPPNNP residues
Synthetic proteins rescue auxotrophs• transformed synthetic genes into single-gene
knockout E.coli strainso tested for new viability on minimal medium
• Four auxotrophs regained viability
• Verified role of de novo protein versus natural revertant mutation
Potential Biological Functions of de novo Proteins (1)
• Protein has role in novel bypass pathway?
• De Novo protein fails to rescue auxotrophs with deletions at other steps in the biosynthetic pathway
Potential Biological Functions of de novo Proteins (2)
• Altering expression or activity of endogenous protein?
• Overexpression of these ‘multicopy supressors’ can rescue deletions
• De Novo proteins worked even when multicopy supressors were deleted
Potential Biological Functions of de novo Proteins (3)
• Global metabolic alterations (ie cellular stress response)?
• Proteins were folded properly (from circular dichroism spectra)
• Failure of de novo mutant to rescue cell indicates function is sequence-specific
Efficiency of de novo proteins• Proteins not selected by evolution or for
enzymatic function
• Even at 400x higher levels than WT, cells grew slower
• Could not detect activity in assay
Replacement of 0.1% of E.coli Genome• probed ability of de novo sequences to rescue multiple
deletions• only ~400 E.coli genes are essential and (at least) 0.1% of
the genome can be replaced by artificial genes
Results
• de novo proteins can sustain cell growtho 4/27 auxotrophs rescued by de novo proteins
• de novo proteins operate with different pathway than natural proteins
• Slow growth, low levels of protein activity
De Novo Proteins
• Purpose / Significanceo Protein selection by structural strategy (binary code)o novel sequences with no similarity to natural proteins can
compensate for gene deletiono Synthetic biology still in infancy compared to billions of years of
evolution
• Flawso Inclusion of work on replacement of E.Coli genomeo failure to determine any pathways, only disprove alternative
mechanismso Relevance of work questionable