CARACTERÍSTICAS DEL SECTOR RETAIL

The ability to attach a payload to a targeting ligand is a fundamental requirement in the field of molecular imaging. While this is simple to do if both the ligand and payload are small molecules, the problem becomes much more complex when either is a protein. Many chemical approaches to bioconjugation have been developed. However, these techniques are often not efficient, stoichiomet- ric, or site specific. They may also exclude large numbers of potential ligands, such as those with disulfide bonds. Enzymatic bioconjugation techniques ameliorate some of these issues, but are also limited by constraining useable payloads to structures chemically similar to their natural substrates, requiring large fusions that alter the properties of the targeting ligand, or necessitating difficult downstream purifications. Keeping all of these considerations in mind, we set out to create a novel bioconjugation technology that combines the small size and flexibility of chemical techniques with the efficiency and specificity of enzymatic approaches.

We were able to do this by utilizing the transpeptidation activity of S. aureus Sortase A. In the presence of Ca2+, the enzyme recognizes the pentapeptide sequence LPXTG and then catalyzes

the replacement of the glycine residue with any other polypeptide that has an N-terminal glycine. Nearly any molecule, biological or synthetic, can flank the LPXTG peptide or follow the N-terminal glycine of the second molecule, yielding an efficient, site-specific transformation unprecedented in its flexibility. Our novel technique, Sortase-Tag Expressed Protein Ligation (STEPL), harnessed the power of the sortase enzyme by expressing its active domain as part of a larger chimeric protein. The STEPL chimera consisted of, from N- to C-terminus, the protein to be conjugated, the sortase recognition motif LPETG, a flexible linker sequence, the sortase active domain, and a hexahistidine tag. The construct was then expressed in the E. coli cytoplasm and purified by metal affinity chromatography. However, rather than eluting the chimeric protein off of the metal resin, the bound beads were incubated with CaCl2and a short, synthetic peptide. This activated the sortase domain,

cleaving the target protein off of the column via conjugation of the peptide. In theory, linking protein cleavage to bioconjugation should result in complete bioconjugation of all target protein that is released from the beads, eliminating the need for large excesses of synthetic peptide and greatly simplifying downstream workups.

In practice, the system works in much the same way. The major difference is due to a peptide- independent hydrolytic shunt in the sortase mechanism. To quantify the effect of this side reaction, a simple test system was constructed using the reporter protein EGFP as the target protein to be conjugated and triglycine as the synthetic peptide. We were able to monitor STEPL activity under a number of initial conditions by measuring the EGFP concentration in the supernatant, which would only occur if the enzyme actively cleaved the protein off of the beads. First, we used this assay to determine the optimal CaCl₂ concentration. While the hydrolytic shunt is active in the absence of peptide, calcium is necessary even for this side reaction. By titering CaCl₂ in the presence and absence of 25 µM triglycine, we calculated a ratio of ligated to unligated free EGFP as a function of calcium concentration. In less than 100 µM CaCl₂, the system reported a high percentage of conjugated product. This ratio quickly dropped off at higher concentrations, so 100µM was determined to be the optimal calcium concentration for the STEPL system.

After that, we used the EGFP-STEPL reporter construct to analyze the effects of reaction time, reaction temperature, and peptide concentration. These parameters are not independent of one another, so a mass-action kinetics model was developed that used the Arrhenius definition of a rate constant to capture temperature-dependence. The model was fit to wet-lab quantification of EGFP

Section 6.1: Thesis Discussion 139

release under varying conditions to determine the pre-exponential constants and activation energies of both the glycine-dependent and glycine-independent pathways of the sortase mechanism. The calculated parameters had reasonable magnitudes and model-predicted EGFP release fit the data well. The model was finally used to predict the yield of conjugated protein, the percentage of re- leased protein that would be conjugated, and the percentage of peptide consumed by the STEPL reaction. Conjugation percentage was heavily dependent on initial peptide concentration and weakly temperature dependent, with more peptide and higher temperatures leading to more pure results. A ratio of initial peptide to initial chimera that gave >95% conjugated protein could be found for all temperatures and times greater than 30 minutes. For a 6 hour reaction, only a two-fold excess of peptide was needed for 95% purity at 37◦C. Peptide utilization was heavily dependent on both tem- perature and initial peptide concentrations. The peptide dependence was the inverse of conjugation purity, with peptide utilization increasing with decreasing initial peptide concentrations. Although peptide utilization of>90% were possible, this only occurred within 6 hours at 37◦C. Finally, overall yield increased with temperature and peptide concentration. Thus, 37◦C was determined to be the optimal temperature for all outcomes. Interestingly, peptide utilization and conjugate purity were found to be competing interests. Therefore, STEPL reactions can be optimized for peptide utiliza- tion if downstream applications facilitate easy separation of conjugated and unconjugated protein, such as subsequent surface functionalization, or for high purity if further purifications would be difficult, for instance if the protein will be used directly for imaging or therapy.

This novel system for protein purification and bioconjugation offers many advantages over other chemical and enzymatic ligation technologies by combining their advantages and avoiding their shortcomings. Like chemical crosslinkers, but unlike most enzymes, STEPL can conjugate a broad selection of bio-orthogonal functionality to a protein while adding minimal bulk. However, being an enzymatic reaction, it is more efficient, site-specific, and quantitative, unlike most chemical conju- gations. Because STEPL consists of a single chimeric protein, it eliminates the need for subsequent purification of the sortase enzyme from the ligation products. What makes STEPL truly unique in the field of bioconjugation, however, is that it links protein purification to bioconjugation. In doing this, it can produce a high-purity, quantitatively labeled product or utilize most of an expensive reagent. Both approaches reduce the time, cost, and complexity of bioconjugation, making it a significant advance in the field with applications throughout academia and industry. STEPL has

the potential to aid in the production of protein-drug conjugates, PEGylated biologics, biophysical probes, and of course molecular imaging agents, among many other applications.