6.2
Future Directions
6.2.1
Extending STEPL to Other Expression Systems
An obvious next step in the development of STEPL is to extend it to other prokaryotic and eukaryotic expression systems. While the E. coli cytoplasm is a convenient and scalable place to express proteins, it is severely limited in the types of proteins that can be expressed. scFvs for example, have much more complex structures than affibodies and often contain multiple disulfide bonds, which cannot form in the reducing environment of the cytoplasm. When overexpressed proteins are poorly folded, they can precipitatein vivo, forming occlusion bodies that complicate harvesting proteins [268]. There are a few strategies for avoiding these precipitates, but the problem is usually best solved by changing expression systems entirely.
The simplest change is to express the STEPL chimera in the bacterial periplasm, which is an oxidizing environment that has been reported to be a favorable compartment for scFv expression [269]. This is accomplished by fusing the OmpA or PelB signaling peptide to the N-terminus of the chimera, before the targeting ligand. The peptide facilitates transport across the inner mem- brane before being proteolyzed, resulting in the original STEPL chimera. During phage display, the STEPL-pIII protein was transported to the periplasm via the OmpA signal peptide. Phage displaying the sortase enzyme showed activity, which is a good indication that periplasmically ex- pressed STEPL chimeras will also remain functional. We have already begun working on expressing scFv-STEPL chimeras in the periplasm. The STEPL chimera has been cloned into the pET20(+) expression plasmid, which directs periplasmic expression via the PelB signal. Initial experiments resulted in poor yield of scFv fusion proteins, so the project has been delegated to the experts at the Wistar Institute’s Protein Expression Facility.
When expressing complicated proteins, however, the best options are eukaryotic. Yeast, insect, and mammalian cells all have a secretory system capable of folding and secreting complex proteins with a myriad of post-translational modifications [270]. Yeast are particularly well suited for protein expression because they can be grown to high densities and secrete the protein for easy isolation and continuous manufacturing [271]. Although they grow slowly compared to bacterial cells, they outpace other eukaryotic systems by a considerable amount. Currently, a collaborator in the Muzykantov group at Penn is working to optimize expression of an scFv-STEPL fusion in theP. pastoris yeast
strain. By optimizing STEPL in eukaryotic expression systems, its utility would grow enormously as it could then be capable of expressing and conjugating full-length antibodies and enzyme-derived therapeutic proteins.
6.2.2
Improved Directed Evolution Strategies
While the directed evolution attempts described in Chapter5were unable to produce sortase enzymes with reduced background cleavage, this will still be necessary to accelerate adoption of the technology throughout the scientific community. To that end, there are a few potential options for improving the directed evolution strategy, depending on what the problem with the current system actually is. In Section5.5.4we altered the screening protocol in an attempt to solve one potential problem, that SBP-streptavidin binding affinity may be too low, resulting in random release of inactive phage. Another solution to that problem is to change the affinity ligand from SBP to one with a stronger interaction with its target. We are already in the process of doing this, cloning a phagemid that displays a hexahistidine tag rather than SBP. The tight binding of His6 with immobilized metal resins is well documented [272, 273, 274, 275]. Even in this thesis, we have shown that a cobalt resin is capable of retaining protein for a full 24 hours. Using a His-tag for immobilization may also reduce the potential to evolve a binding pocket for the affinity ligand because it is much shorter than SBP and lacks any sort of structure necessary for protein-protein interactions.
Another way to avoid evolving an affinity ligand binding pocket is to use larger ligands, such as monobodies or nanobodies. Even if a modulated, tight binder were to emerge, it would have a much lower chance of binding the ligand in a configuration that impairs binding to an immobilized target. To further ensure this, several copies of the ligand could be strung together one after another. Not only would that greatly increase the overall avidity to the plate, it would also leave extra ligands that are capable of binding the plate even if one of them was sequestered by an evolved binding loop, making it much more difficult for that phage to pass the screen.
Phage display itself may be too complicated of a system for the delicate evolution of an enzyme. If that is the case, a delicate screening system such as ribosome display may be more appropriate. Ribosome display is entirely in vitro, so it removes many possible complications, such as STEPL- phage interactions, the oxidizing environment of the periplasm, and transport across the inner membrane. It would also allow much larger libraries that could increase the possibility of recovering clones with the desired phenotype.