Production of a toxic polypeptide as a fusion inside GroEL cavity

Maria S Yurkova,Elchin G Sadykhov,Alexey N Fedorov

doi:10.1038/s41598-020-78094-8

Abstract

The system is developed for efficient biosynthetic production of difficult-to-express polypeptides. A target polypeptide is produced fused into T. thermophilus GroEL chaperonin polypeptide chain in such a way that it is presented inside the GroEL cavity near the substrate binding surface. Such presentation allows alleviating potential problems of instability, toxicity or hydrophobicity of the fused peptide. Thermostability of thermophilic GroEL can be used for its one-step separation from the host cell proteins by heating. The target polypeptide may be released by any of amino acid-specific chemical treatments. In this study, GroEL was adapted for methionine-specific cleavage with cyanogen bromide by total replacement of methionine residues to facilitate further purification of the target polypeptide. The procedure is simple, robust and easy to scale-up. The capacity of this system to produce difficult-to-express polypeptides is demonstrated by production in bacterial system of one of the most potent antibacterial peptides polyphemusin I.

Highlights

The system is developed for efficient biosynthetic production of difficult-to-express polypeptides
This study describes the development of a GroEL-based fusion carrier, where a target polypeptide is inserted into the chaperonin polypeptide chain so as to be presented inside the cavity near its substrate-binding surface, while the oligomer structure of the particle is not hampered
GroEL from T. thermophilus has six methionine residues in its sequence; the gene construct encoding GroEL with all methionine residues replaced by leucine residues was obtained by PCR with appropriate primers

Summary

Introduction

The system is developed for efficient biosynthetic production of difficult-to-express polypeptides. A target polypeptide is produced fused into T. thermophilus GroEL chaperonin polypeptide chain in such a way that it is presented inside the GroEL cavity near the substrate binding surface Such presentation allows alleviating potential problems of instability, toxicity or hydrophobicity of the fused peptide. The main purpose of our work was to develop a widely applicable fusion protein system that would be suitable for the production of different peptides which are difficult to express in bacteria for the reasons described above, and that would allow simple, robust and scalable purification procedures of target polypeptides. The chaperone GroEL is a fitting candidate to be used as a carrier in such a fusion system[18] It comprises a large particle consisting of two heptamers with a large cavity inside, which accommodates the protein substrate. It is known that random transposon inserts usually inactivate proteins and influence their native structure

Objectives

Methods

Results

Conclusion