Tailored Polyproteins Using Sequential Staple and Cut

Abstract

Polyproteins, individual protein units joined covalently in tandem, have evolved as a promising tool for measuring the dynamic folding of biomacromolecules in single-molecule force spectroscopy. However, the synthetic routes to prepare polyproteins have been a bottleneck, and urge development of in vitro methods to knit individual protein units covalently into polyprotein. Employing two enzymes of orthogonal functionalities periodically in sequence, we synthesized monodispersed polyproteins on a solid surface. We used Sortase A (SrtA), the enzyme known for sequence specific transpeptidation, to staple protein units covalently through peptide bonds. Exploiting the sequence-specific peptide cleaving ability of TEV protease, we controlled the progress of the reaction to one attachment at a time. Finally, with unique design of the unit proteins we control the orientation of proteins in polyprotein. This simple conjugation has the potential to staple proteins with different functionalities and from different expression systems, in any number in the polyprotein and, above all, via irreversible peptide bonds. Multiple chimeric constructs can also be synthesized with interchangeable protein units.

Structural basis of the strong cell‐cell junction formed by cadherin‐23

“…We used Cdh23 EC1‐2 and Cdh23 EC1‐3 for the experiment and not Cdh23 EC1, as Cdh23 EC1 showed the weakest affinity toward dimerization in the SEC. Since we performed smFRET on a glass coverslip using a total internal reflection fluorescence microscopy (TIRFM), we covalently anchored the C terminus of the proteins, Cdh23 EC1‐2, and Cdh23 EC1‐3 individually, to the coverslips using sortagging chemistry as reported . All protein constructs were recombinantly modified with the sortase recognition sequence (–LPETGSS) at the C terminus.…”

Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level

“…Nevertheless, these results are already an advance compared with other ligase-based protein ligation method. Previous protein ligase-dependent polymerization seldom reported the construction of a protein pentamer, and the yield for a protein tetramer was <1% at the single-molecule level 12,55 . Thus, our method provides a tool for biotechnology and protein engineering, which is suitable for both protein coupling and immobilization.…”

“…There are other enzymatic methods which are well-suited for linking protein as a dimer or for protein labeling, such as sortase, split intein, bultease, and SpyCather-SpyTag 12,55–57 . Sortase is another similar peptidase which is promising for building long protein polymer.…”

“…A modified method was later developed consisting of a reducing-resist thiol-ether bond formation using maleimide-thiol chemistry 64 . Recently, similar ligase-based polymerization methods using sortase were also developed 12,55 . All these methods enable the pre-purification of protein monomer before polymerization.…”

Next Generation Methods for Single-Molecule Force Spectroscopy on Polyproteins and Receptor-Ligand Complexes

“…The Sortase system exhibits a high micromolar K m value, requiring high concentrations of the substrates. This system has been used for AFM-SMFS for immobilization of protein directly from cell lysate (Srinivasan et al, 2017) or in systems where an LPETGG tag and GGG tag have been used to assemble polyproteins posttranslationally or to attach high-strength Dockerin handles to proteins (Durner et al, 2017;Garg et al, 2018;Liu et al, 2018).…”