(331c) Bacterial Inner Membrane Display to Simultaneously Engineer Solubility and Antigen Binding of Intracellular Antibodies

Intracellular antibodies, antibodies that fold and function inside cells, present a powerful research tool for inhibiting and redirecting protein function. However, engineering intrabodies is challenging, because the antibodies must fold in the reducing intracellular environment without the disulfide bonds normally required for proper folding. To overcome this challenge, we have developed an Escherichia coli inner membrane display method that utilizes a translocation intermediate of the twin-arginine translocation (Tat) pathway. Proteins targeted for transport by the Tat pathway must fold in the cytoplasm prior to translocation to the periplasm, due to the folding quality control feature of the Tat pathway that prevents translocation of poorly folded proteins. During translocation, an intermediate forms with the N-terminus of the translocating protein anchored to the inner membrane and the C-terminus of the protein in the periplasm. This intermediate can be detected on the inner membrane and probed for binding to a target protein in solution or coated on beads. Since the Tat machinery checks for cytoplasmic folding prior to transport and display, only well folded proteins are displayed on the inner membrane and interrogated for binding to a target antigen, which eliminates the need for separate screening steps to engineer solubility and antigen binding. We used our technique to display and screen a combinatorial single-chain variable fragment (scFv) antibody library based on scFv13, resulting in simultaneous improvement of solubility and antigen binding. Following these proof-of-concept experiments, we displayed a naïve scFv library in E. coli and used our technique to isolate and affinity mature soluble scFvs for survivin, a biologically relevant target in cancer cells. We expressed the engineered scFvs in a mammalian cell line to demonstrate their solubility and potential for intracellular applications. Our results highlight the applicability of our display technique for rapid isolation of high-affinity, soluble intracellular antibodies.