Conceptual and methodological advances in cell-free directed evolution

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Highlights

  • Advances in cell-free evolution allow for more sophisticated protein engineering.

  • New display formats enable evolution of multimeric and integral membrane proteins.

  • Incorporation of unnatural amino acids expands chemical repertoire in vitro.

  • Technical enhancements improve method accessibility, efficiency, and robustness.

Although cell-free directed evolution methods have been used to engineer proteins for nearly two decades, selections on more complex phenotypes have largely remained in the domain of cell-based engineering approaches. Here, we review recent conceptual advances that now enable in vitro display of multimeric proteins, integral membrane proteins, and proteins with an expanded amino acid repertoire. Additionally, we discuss methodological improvements that have enhanced the accessibility, efficiency, and robustness of cell-free approaches. Coupling these advances with the in vitro advantages of creating exceptionally large libraries and precisely controlling all experimental conditions, cell-free directed evolution is poised to contribute significantly to our understanding and engineering of more complex protein phenotypes.

Introduction

Directed evolution is a powerful tool for tailoring biomolecular properties. For applications involving proteins, the approach requires a one-to-one mapping of phenotype and genotype, since selections or screens are performed on the proteins themselves, but amplification and identification of the desired sequences need to be performed at the genetic level. This phenotype–genotype linkage is easily achieved in cells, which can naturally compartmentalize individual DNA sequences and, through transcription and translation, the corresponding proteins. However, the use of cells significantly reduces the size of libraries that can be sampled, which can be vital, particularly when biomolecular functions are evolved completely de novo; additionally, cell-based directed evolution limits the scope of buffers, solvents, and temperatures that can be used since cell viability must be maintained.

In 1997, the first truly cell-free directed protein evolution platforms  mRNA display [1] and ribosome display [2]  were developed as iterative procedures that replicate the natural evolutionary processes of mutation, selection, and amplification (Figure 1). The field of cell-free protein engineering has rapidly expanded over the past two decades, with notable advances in the use of defined translation mixtures [3], engineering of cell-toxic or aggregation-prone proteins [4], broadening selection and screening assays [5], and optimization of selection conditions and parameters [6]. By leveraging these advances and the unique evolutionary context provided by truly cell-free selections, researchers have also begun to investigate the origins of life [7]; in the context of structural biology, cell-free directed evolution provides a means for studying how primordial biomolecules evolved to adopt three-dimensional conformations that engendered novel functions [8]. Many well-established applications of cell-free protein engineering approaches are comprehensively reviewed elsewhere [9, 10]. Here, we focus on conceptual and methodological advances in cell-free directed protein evolution over the past few years, and we offer our perspective on the promising future directions of the field.

Section snippets

Conceptual advances that have expanded the scope of selectable molecular phenotypes

Some of the most common molecular phenotypes that cell-free protein engineers seek to create include high-affinity binding against cellular targets (ideally with high target specificity), more efficient biocatalysis (often paired with improved or altered substrate specificity), and higher protein stability under harsh solvent conditions. Such phenotypes have typically been evolved from monomeric, water-soluble protein templates. While the experimental focus on monomeric proteins can be

Methodological advances that have improved accessibility, efficiency, and robustness of cell-free approaches

Beyond conceptual innovations widening the breadth of problems that in vitro selections can address, recent methodological advances have enhanced the accessibility, efficiency, and robustness of existing cell-free selection schemes (Figure 3). These improvements set the stage for reliable methods that researchers can readily implement to carry out cell-free directed evolution.

Accessibility of cell-free technologies to new researchers remains a challenge, since complete cell-free directed

Conclusion and outlook

Combinatorial cell-free protein engineering continues to be an active, quickly-developing field, providing the research community established and novel tools for creating and improving biomolecular function. When biomolecular engineering projects rely on either sampling the largest possible libraries or exerting full control over the experimental selection conditions, in vitro display methods are clearly favored over cell-based schemes. Here we have reviewed recent work highlighting the

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by grants from the National Institutes of Health (CA179180) and the National Science Foundation (CBET-1055231) to C.A.S.

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