Strategies for in vitro engineering of the translation machinery

Abstract

Engineering the process of molecular translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chemical biology to generate novel biological insights and enable new applications (e.g. designer protein therapeutics). Here, we review methods for engineering the process of translation in vitro. We discuss the advantages and drawbacks of the two major strategies-purified and extract-based systems-and how they may be used to manipulate and study translation. Techniques to engineer each component of the translation machinery are covered in turn, including transfer RNAs, translation factors, and the ribosome. Finally, future directions and enabling technological advances for the field are discussed.

Figure 1.

Conceptual goals for engineering templated polymer production by the ribosome. (A) The standard genetic code enables the polymerization of canonical amino acids with a diversity of 20 proteinogenic side chains (blue shaded circles). Despite enabling the evolution of life and having been harnessed for societal needs (e.g. recombinant protein production of protein therapeutics like insulin), protein biosynthesis in nature uses limited sets of protein monomers, which results in limited sets of biopolymers (i.e. proteins). (B) First-generation genetic code expansion facilitates the incorporation of L-α-amino acids with a vast array of chemical side chains into proteins (pink circles). Site-specific incorporation of up to 40 instances of a single ncAA in a single polypeptide chain has been reported (10). (C) Next-generation genetic code expansion involves the incorporation of monomers with both non-canonical side chains and backbones (multi-colored stars). Engineering of all aspects of the translation apparatus will be required to generate systems capable of efficiently carrying out polymerization of these exotic new molecules.

Figure 2.

In vitro protein synthesis systems facilitate translation system engineering. Two strategies exist for enabling protein translation in vitro: the PURE system and extract-based systems. In the PURE system (left), each unique component of the translation apparatus is individually purified from cells, including the aminoacyl-tRNA synthetases, tRNAs, translation factors, and ribosomes. In order to reconstitute a functional translation system, these components are then recombined together with amino acids, energy substrates, cofactors, salts, a template for a protein of interest (POI), and T7 RNA polymerase (RNAP) to generate mRNA template. Importantly, this methodology enables precise optimization of component concentrations and the ability to leave out certain components and replace them with modified components to modulate translation apparatus function. In contrast, extract-based systems (right) entail a simpler protocol for the preparation of a crude cellular extract containing all the necessary components.

Figure 3.

tRNA aminoacylation methods for non-canonical amino acid incorporation. Methods for tRNA aminoacylation may be divided into two categories – those which leverage engineered orthogonal variants of the protein aminoacyl-tRNA synthetases (o-AARS) used by organisms to charge tRNAs in cells, and those which bypass this system via alternative routes. Systems using an o-AARS/o-tRNA pair (left) follow one of two methodologies. In the first, the o-AARS and o-tRNA are individually purified and may be added at any desired concentration to either a PURE reaction or an extract-based reaction. If fewer purification steps are desired, the o-AARS/o-tRNA pair maybe expressed in cells from which an extract is directly prepared, alleviating the need to supplement it in the reaction, but ceding some control of reaction conditions. Alternative routes (right) are often used for monomers which do not have engineered o-AARS variants available. The first involves T4 ligase-mediated ligation of an aminoacylated pdCpA to a truncated tRNA (right-left). The second avoids the challenging ligation step by chemically modifying a monomer which is already aminoacylated to a tRNA by a native AARS (right-center). Lastly, artificial ribozymes called Flexizymes may be utilized to aminoacylate tRNAs with a wide range of non-canonical amino acids and other monomers (right-right). Once obtained and purified, these aminoacylated tRNAs may be used readily in a PURE or extract-based method for translation. Strain engineering or selective depletion can be used to modify the content of translational components in the extract.

Figure 4.

Engineering translation system components. tRNA (center-top) and ribosome (center-bottom) regions are labeled by name, and segments/ nucleotides that are known to be mediate specific processes of translation are labeled and color-coded by the translation factors they interact with. The most commonly engineered translation factors are depicted and labeled with regions of the molecule that may be targeted to modulate function.

Figure 5.

Generation of ribosomal variants for engineering the translation machinery in vitro. Ribosome libraries can be generated by two main strategies. In one approach, ribosome libraries are built in vivo and entail transformation of a library of rRNA variants, expression of those variants in living cells, and purification of fully assembled ribosomes for in vitro manipulation (top). One drawback of this method is that dominant lethal genotypes—those which kill any cell in which they are expressed—will not be present in the final library (grayed out cells). In contrast, methods for building ribosomes purely in vitro can avoid this constraint, enabling the construction of many ribosomal variants which may be lethal in vivo (purple). It is possible, however, that this library may be missing ribosomal variants which have difficulty assembling properly in vitro (yellow).