de novo designed proteins from a library of artificial sequences function in escherichia coli and...
TRANSCRIPT
![Page 1: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/1.jpg)
De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and
Enable Cell Growth
20.385March 7, 2012
Hannah Johnsen and Sabina Sood
Michael A. Fisher, Kara L. McKinley, Luke H. Bradley, Sara R. Viola, Michael H. Hect
![Page 2: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/2.jpg)
Background
• De novo - starting from the beginning, from scratch
• Binary code strategy - specific sequence pattern of polar and non-polar residues
• Four-helix bundle - four helices packed in a coiled-coil arrangement
• Auxotroph - unable to synthesize compounds required for growth
![Page 3: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/3.jpg)
Overview
• Purpose: Determine if de novo proteins can replace growth function in cells
•I. Design of novel proteins
•II. Rescue by de novo proteins
•III. Binary pattern design
•IV. Testing of E. coli strains
•V. Rescue of knockout E. coli
![Page 4: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/4.jpg)
Design of novel proteins
Figure 1: Design of a collection of novel proteins and rescue of E. coli auxotrophs.
Red: Polar residue
Yellow: Non-polar residue
![Page 5: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/5.jpg)
Rescue by de novo proteins
Figure 2. Rescue of E. coli auxotrophs by de novo proteins
![Page 6: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/6.jpg)
Binary pattern design
• Four auxotrophs were able to be rescued:
1. serB
2. gltA
3. ilvA
4. fes
Figure 3. Designed amino acid sequences that enable growth of E. coli auxotrophs
![Page 7: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/7.jpg)
Biological functions of de novo proteins
• serB: phosphoserine phosphatase
• gltA: citrate synthase
• ilvA: threonine deaminase
• fes: enterobactin esterase
![Page 8: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/8.jpg)
Verification of de novo proteins
• Auxotroph survived by mutationo New auxotrophs transformed o Saw similar growth
• Auxotroph survived by uptake of other plasmid DNAo Isolated sequenceo Recloned into new vector
![Page 9: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/9.jpg)
Testing of E. coli strains
Figure 4. Growth of auxotrophic strains of E. coli in selective liquid media
![Page 10: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/10.jpg)
Possible mechanisms for rescue
• 1. Encode bypass pathways:o De novo sequences transformed into cells with
enzyme deletiono Discovered: sequences did not rescue cells
•2. Alter expression or activity of endogenous protein:
o Screen to identify overexpression of natural geneso Transformed double deletion strainso Discovered: novel sequences rescue double
deletions
![Page 11: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/11.jpg)
• 3. Cause unfolded sequences that induce a stress response:o Purified proteins and measured circular dichroism
spectrao Discovered: structures are predominantly alpha-
helical
Possible mechanisms for rescue
![Page 12: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/12.jpg)
Possible mechanisms for rescue
• Do mediate rescue of specific chromosomal deletions
• Do rescue expression by sequence-specific features
![Page 13: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/13.jpg)
Rescue of knockout E. coli
Figure 5. Rescue of a quadruple knockout E. coli by co-expression of 4 de novo proteins
![Page 14: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/14.jpg)
Concerns
• De novo protein showed very low levels of protein activity
• De novo proteins were not specifically engineered, just random library
• Never mentioned how the de novo proteins rescue the auxotrophs
![Page 15: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/15.jpg)
Conclusions
• Sequences designed de novo can provide necessary functions for growth
• Cell growth can be sustained by simpler structures
• De novo proteins exhibit lower levels of biological activity
![Page 16: De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth 20.385 March 7, 2012 Hannah Johnsen](https://reader031.vdocuments.us/reader031/viewer/2022032205/56649e7e5503460f94b80cf4/html5/thumbnails/16.jpg)
Significance
• Toolkit for synthetic biology is no longer limited to genes and proteins that already exist in nature
• Could lead to novel evolutionary trajectories
• Future work: Initial step towards the construction of artificial genomes