Synthetic Biology Approaches to Engineering Human Cells

Conditional protein splicing is a powerful biotechnological tool that can be used to rapidly and posttranslationally control the activity of a given protein. Here we demonstrate a novel conditional splicing system in which a genetically encoded protein scaffold induces the splicing and activation of an enzyme in mammalian cells. In this system the protein scaffold binds to two inactive split intein/enzyme extein protein fragments leading to intein fragment complementation, splicing, and activation of the firefly luciferase enzyme. We first demonstrate the ability of antiparallel coiled-coils (CCs) to mediate splicing between two intein fragments, effectively creating two new split inteins. We then generate and test two versions of the scaffold-induced splicing system using two pairs of CCs. Finally, we optimize the linker lengths of the proteins in the system and demonstrate 13-fold activation of luciferase by the scaffold compared to the activity of negative controls. Our protein scaffold-triggered conditional splicing system is an effective strategy to control enzyme activity using a protein input, enabling enhanced genetic control over protein splicing and the potential creation of splicing-based protein sensors and autoregulatory systems. ■ INTRODUCTION Protein splicing is a post-translational modification that can control the activity of a protein by assembling it from inactive fragments. Analogous to RNA splicing, protein splicing is the process by which an intervening protein domain, or intein, selfexcises out of a larger polypeptide, ligating the two flanking polypeptidestermed exteinsinto a single protein. Protein splicing can occur in cisor in trans-. 2−4 For trans-splicing the intein sequence is split into two fragments, and the splicing reaction occurs between two distinct polypeptides. Intein domains are highly modular and have been used by researchers to assemble several different proteins in various experimental contexts.5−8 The development of conditional protein splicing has enabled researchers to post-translationally control protein activity in response to specific molecular inputs and has already become a useful research tool. By engineering split inteins that contained the rapamycin ligand-binding domains, Mootz et al. first demonstrated that trans-splicing could be induced by the small molecule rapamycin. Other conditional splicing systems have been generated to induce splicing in response to temperature, light, and chemical ligands such as 4-HT.9−13 Here we propose a novel conditional splicing system in which splicing is induced by the presence of a genetically encoded protein scaffold. In contrast to previously developed conditional splicing systems activated by exogenously administered inducers (small molecules and light), a protein inducer can be directly linked to endogenous biological pathways. This characteristic enables the potential to monitor or rewire biological pathways at the protein level. The engineering of synthetic post-translational signaling pathways reflects a common strategy used by natural systems and is a major research focus. However, only a few general approaches exist.14−18 The protein-induced protein splicing system offers a general mechanism to post-translationally convert a protein input into a fully formed output protein through engineered binding domains. The general reaction mechanism demonstrated by our work also suggests methods to create splicingbased protein sensors and autoregulatory systems. Biomolecular scaffolds have previously been used to increase the yield of biochemical synthesis pathways by bringing together enzymes operating in a pathway.19−22 We hypothesized that protein scaffolds could also be used to bring together split inteins and trigger protein splicing. Our system consists of two fusion proteinseach containing split intein/extein domains fused to a scaffold binding domainand the input scaffold protein. In the presence of the scaffold the two fusion proteins bind to the scaffold leading to association of the intein fragments, splicing, and activation of the output protein. We constructed, tested, and optimized a scaffold-induced splicing system comprising well-characterized protein components. We used two pairs of previously characterized antiparallel coiled-coils termed LZA/LZB and EE/RR.23−25 LZA is known to bind strongly to LZB and EE to RR, but no binding is expected to occur between proteins in the different pairs. These coiled-coils drive the specific association of the intein/extein fusion proteins and the synthetic scaffold. We Received: February 15, 2013 Article pubs.acs. org/JACS © XXXX American Chemical Society A dx.doi. org/10.1021/ja401689b | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX