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Continuous directed evolution of proteins with improved soluble expression

Abstract

We report the development of soluble expression phage-assisted continuous evolution (SE-PACE), a system for rapidly evolving proteins with increased soluble expression. Through use of a PACE-compatible AND gate that uses a split-intein pIII, SE-PACE enables two simultaneous positive selections to evolve proteins with improved expression while maintaining their desired activities. In as little as three days, SE-PACE evolved several antibody fragments with >5-fold improvement in expression yield while retaining binding activity. We also developed an activity-independent form of SE-PACE to correct folding-defective variants of maltose-binding protein (MBP) and to evolve variants of the eukaryotic cytidine deaminase APOBEC1 with improved expression properties. These evolved APOBEC1 variants were found to improve the expression and apparent activity of Cas9-derived base editors when used in place of the wild-type cytidine deaminase. Together, these results suggest that SE-PACE can be applied to a wide variety of proteins to rapidly improve their soluble expression.

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Fig. 1: Overview of a PACE selection for improved soluble protein expression.
Fig. 2: Validation of the soluble protein expression PACE selection.
Fig. 3: Split-intein pIII as an AND gate for bridging two positive selections in PACE.
Fig. 4: Soluble expression PACE with simultaneous selection for expression and binding activity.
Fig. 5: Activity-independent selection for soluble proteins in PACE.

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Acknowledgements

This work was supported by US NIH NIBIB R01EB022376, NIGMS R35 GM118062, NHGRI RM1 HG009490, Monsanto, and the Howard Hughes Medical Institute. T.W. is a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellow (F32 GM119228).

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T.W. designed the research, performed the experiments, analyzed data, and wrote the manuscript. A.H.B. designed the research, analyzed data, and wrote the manuscript. T.P.H. performed and analyzed the mammalian cell base editing experiments. D.R.L. designed and supervised the research and wrote the manuscript.

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Correspondence to David R. Liu.

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The authors have filed a provisional patent application on the PACE system and related improvements.

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Wang, T., Badran, A.H., Huang, T.P. et al. Continuous directed evolution of proteins with improved soluble expression. Nat Chem Biol 14, 972–980 (2018). https://doi.org/10.1038/s41589-018-0121-5

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