Sortase-Tag Expressed Protein Ligation: Combining Protein Purification and Site-Specific Bioconjugation into a Single Step

Abstract

Efficient labeling of protein-based targeting ligands with various cargos (drugs, imaging agents, nanoparticles, etc.) is essential to the fields of molecular imaging and targeted therapeutics. Many common bioconjugation techniques, however, are inefficient, non-stoichiometric, not site-specific, and/or incompatible with certain classes of protein scaffolds. Additionally, these techniques can result in a mixture of conjugated and unconjugated products, which are often difficult to separate. In this study, a bacterial sortase enzyme was utilized to condense targeting ligand purification and site-specific conjugation at the C-terminus into a single step. A model was produced to determine optimal reaction conditions for high conjugate purity and efficient utilization of cargo. As proof-of-principle, the sortase-tag expressed protein ligation (STEPL) technique was used to generate tumor-specific affinity ligands with fluorescent labels and/or azide modifications at high purity (>95%) such that is was not necessary to remove unconjugated impurities. Click chemistry was then used for the highly efficient and site-specific attachment of the azide-modified targeting ligands onto nanoparticles.

Advances in nano-based inner ear delivery systems for the treatment of sensorineural hearing loss

“…Bioconjugation allows for the creation of stable peptide bonds, thus anchoring and immobilizing ligands to the nanoparticle surface. Many bioconjugation techniques have been described in the literature including maleimide, N-hydroxysuccinimide, and carboiimide-based chemistries [57], but few have been found to consistently and effectively add ligands with the correct orientation, surface density, and site-specificity [58,59]. We will briefly review two more recently developed techniques, expressed protein ligation (EPL) and click chemistry.…”

Recent Advances in the Generation of Antibody–Nanomaterial Conjugates

“…[114] Despite these obvious advantages, there are reports showing limitations for the enzyme-based coupling approaches, which arise mainly by fusion proteins being too large and preparation procedure to undertake the coupling reaction being inefficient due to multiple steps. [115] Leung et al developed an alternative strategy to overcome some of these limitations using sortase A enzyme (Srt A) (Figure 13). These enzymes specifically recognize the leucineproline-glutamic acid-threonine-glycine (LPETG) peptide motifs in the protein substrate, which will be cleaved and modified to form a stable amide bond.…”

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

“…In 2013, a particularly creative SrtA-based conjugation strategy was developed to reduce the number of purification steps required to isolate a final conjugate. In this approach, a fusion protein is created in which the protein of interest is attached to a (His) 6 -tagged variant of SrtA by a flexible linker that includes the LPXTG recognition motif [29]. After the immobilization of the protein-LPXTG-linker-SrtA-(His) 6 construct on a solid support, Ca 2+ and a GGG-tagged cargo are added, prompting the site-specific modification of the protein of interest and the consequent release of the protein-cargo conjugate, ultimately leaving the His 6 -tagged SrtA enzyme attached to the solid support.…”