GroEL/ES mediated the in vivo recovery of TRAIL inclusion bodies in Escherichia coli

Zhanqing Wang,Min Zhang,Jing Sun,Jiying Fan,Xin Lv,Jian Zhang,Yaling Shen

doi:10.1038/s41598-018-34090-7

Abstract

Inclusion body (IB) formation generates substantial bio-waste in the pharmaceutical industry and remains a major challenge for heterologous protein expression. Although chaperones can be co-expressed to improve soluble protein yield, their contribution to IB processing in vivo has not been thoroughly studied. Here, a GroEL-GroES co-expressing strain and a deficient strain were constructed to study the in vivo recovery of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The interaction between GroEL/ES and TRAIL was simulated by molecular docking and identified by co-immunoprecipitation. The in vitro cytotoxicity of TRAIL IBs before and after in vivo recovery was subsequently determined by MTT assay. Additionally, IB structures were measured by Fourier transform infrared (FT-IR) spectroscopy and fluorescence spectroscopy. The results showed that after in vivo refolding, IBs retained lower levels of anti-tumor activity and fewer native-like β-sheet structures. Fewer recoverable polypeptides were trapped in IBs after GroEL/ES co-expression and refolding in vivo. Therefore, GroEL/ES mediated the in vivo recovery of TRAIL IBs in Escherichia coli. These results may identify potential uses for IBs and provide additional insight into the detailed mechanisms of in vivo protein recovery.

Highlights

Inclusion bodies (IBs) are frequently encountered in biochemical and biotechnological research[1,2]
The results show that GroEL/ES was important for both the growth of engineered strains and Recombinant human TRAIL (rhTRAIL) expression
To measure the cytotoxic activity of different Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) IBs produced by engineered strains, we studied the inhibition of cell proliferation using the tumor cell line NCI-H460 by using an MTT assay

Summary

Introduction

Inclusion bodies (IBs) are frequently encountered in biochemical and biotechnological research[1,2]. IBs can act as temporary storage for aggregate-prone polypeptides, and heterologous proteins undergo refolding in vitro and in vivo following initial incorrect folding in Escherichia coli[6]. Molecular chaperones are assumed to be associated with soluble TRAIL production, this hypothesis has not been confirmed. Chaperone co-expression is a promising method to improve recombinant protein production, as molecular chaperones have been reported to be involved in protein folding and in the assembly of a variety of substrate proteins[7]. GroE chaperonins help refold denatured peptides into soluble proteins in vitro. GroEL/ES co-expressing and deficient strains—GroEL/ES+ and GroEL/ES−, respectively—were constructed based on the E. coli strain C600, and the IB quality and interactions between these chaperones and TRAIL during the in vivo recovery process were studied. The results of this study provide additional insights into the relationship between molecular chaperones and IBs

Methods

Results

Conclusion