Conceptual and methodological advances in cell-free directed evolution
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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