Conceptual and methodological advances in cell-free directed evolution

Abstract

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.

Figure 1. In vitro selection schemes for mRNA display and ribosome display

A naïve DNA library is first transcribed into mRNA, for use in either mRNA display or ribosome display. In mRNA display, 3′ puromycin ligation and in vitro translation yield display particles comprised of mRNA and the corresponding translated protein, physically linked by puromycin. In ribosome display, in vitro translation of the mRNA directly yields the display particles, consisting of the mRNA, a stalled ribosome, and the corresponding translated protein. In each case, the display particles are then subjected to a selection assay, such as binding to an immobilized target. The selected particles undergo RT-PCR to yield an enriched DNA library. This enriched library is either transcribed again to go through subsequent in vitro display rounds or analyzed to obtain successfully engineered proteins.

Figure 2. Schematic overview of recent conceptual advances in cell-free macromolecular display

Novel or improved display particles and experimental designs expand the scope of protein phenotypes selectable by in vitro display methods. (a) Monovalent display of multimeric proteins [13**,16]. (b) Multivalent display of monomeric proteins [22,23*,25]. (c) Liposome display of integral membrane proteins [28*]. (d) mRNA display with incorporation of unnatural amino acids [33,34,38**].

Figure 3. General flow diagram of cell-free directed evolution with notable recent advances highlighted

During each round of evolution (black), the DNA library is transcribed to mRNA and then translated under specific conditions to create selectable particles that link genotype and phenotype. These particles undergo a selection or screening step, and recovered particles are converted back into an enriched DNA library, which can be further analyzed or directly input into a new round. Conceptual advances (blue) focus on improving the display particles to allow selection of complex molecular phenotypes, as more specifically illustrated in Figure 2. Methodological advances (red) improve the accessibility, efficiency, and robustness of the distinct processes involved in in vitro directed evolution, and can generally reduce the time required for successful evolution (grey).