Ferritin Conjugates With Multiple Clickable Amino Acids Encoded by C-Terminal Engineered Pyrrolysyl-tRNA Synthetase.

Yi-Hui Wang,Yane-Shih Wang,Jo-Chu Tsou,Pei-Jung Chen,Le P Truong,Mu-Lung Jian

doi:10.3389/fchem.2021.779976

Abstract

This study reports the application of expanding genetic codes in developing protein cage-based delivery systems. The evolved Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)•tRNAPyl pairs derived from directed evolution are examined to probe their recognition for para-substituted phenylalanine analogs. The evolved MmPylRS, AzFRS, harboring a wide range of substrates, is further engineered at the C-terminal region into another variant, AzFRS-MS. AzFRS-MS shows suppression of the elevated sfGFP protein amount up to 10 TAG stop codons when charging p-azido-l-phenylalanine (AzF, 4), which allows the occurrence of click chemistry. Since protein nanocages used as drug delivery systems that encompass multiple drugs through a site-specific loading approach remain largely unexplored, as a proof of concept, the application of AzFRS-MS for the site-specific incorporation of AzF on human heavy chain ferritin (Ftn) is developed. The Ftn-4 conjugate is shown to be able to load multiple fluorescence dyes or a therapeutic agent, doxorubicin (Dox), through the strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction. Aiming to selectively target Her2+ breast cancer cells, Ftn-4-DOX conjugates fused with a HER2 receptor recognition peptide, anti-Her2/neu peptide (AHNP), is developed and demonstrated to be able to deliver Dox into the cell and to prolong the drug release. This work presents another application of evolved MmPylRS systems, whose potential in developing a variety of protein conjugates is noteworthy.

Highlights

Protein cages consist of a self-assembled protein hollow shell, such as viral capsids, virus-like particles, chaperonins, and human ferritin heavy chain (Ftn) (Aumiller et al, 2018)
The mutations at the active site of these three MmPylRS variants cover two conserved mutation sites at N346 and C348, together with randomly distributed mutations at other sites (Table 1; Figure 2B). Both the pCDF vector containing the mutated pyrrolysyl-tRNA synthetase (PylRS) gene and the pET vector containing tRNAPyl and the sfGFP-27am gene are co-introduced into E. coli
AzF (4) and p-propargyl-L-phenylalanine (PrF, 5), from which versatile functional groups can be derived for click reactions such as strain-promoted azide-alkyne cycloaddition (SPAAC) and CuAAC, show noticeable signal enhancement when being charged by AzFRS

Summary

Introduction

Protein cages consist of a self-assembled protein hollow shell, such as viral capsids, virus-like particles, chaperonins, and human ferritin heavy chain (Ftn) (Aumiller et al, 2018). These biological supermolecules formed by multiple copies of monomer possess symmetric threedimensional structures with remarkable stability. Protein cages are widely used as delivery platforms in bio-nanotechnology and material science. In the field of drug delivery, protein cages are employed to prolong the half-life of drugs, exert enhanced permeability and retention effect, reduce nonspecific uptake by introducing targeting ligand, and enhance drug solubility or endocytosis effect (Zhang et al, 2021).

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