Human αB-crystallin as fusion protein and molecular chaperone increases the expression and folding efficiency of recombinant insulin.

Mohsen Akbarian,Reza Yousefi

doi:10.1371/journal.pone.0206169

Abstract

Low expression and instability are significant challenges in the recombinant production of therapeutic peptides. The current study introduces a novel expression and purification system for human insulin production using the molecular chaperone αB-crystallin (αB-Cry) as a fusion partner protein. Insulin is composed of A- and B-chain containing three disulfide bonds (one intarchain and two interchains). We have constructed two plasmids harboring the A- or B-chain of insulin joined with human αB-Cry. This system is suitable for cloning of the genes and for directing the synthesis of large amounts of the fusion proteins αB-Cry/A-chain (αB-AC) and αB-Cry/B-chain (αB-BC). The construction of vectors, their efficient expression in Escherichia coli and simple purification of the fusion proteins and two insulin chains are described. A large amount of the recombinant fusion proteins with high purity was obtained by applying a single step anion exchange chromatography or metal chelate affinity. The insulin A- and B-chain were released from the fusion proteins using cyanogen bromide cleavage. The insulin peptides were obtained with an appreciable yield and high purity using one-step gel filtration chromatography. To increase efficiency of chain combination to produce insulin, αB-Cry was used under oxidative conditions. The purification of natively folded insulin was performed by phenyl sepharose hydrophobic interaction chromatography. Finally, using an insulin tolerance test in mice and various biophysical methods, the structure and function of purified human recombinant insulin was compared with authentic insulin, to verify folding of insulin to its native state. Overall, the novel expression system using αB-Cry is highly demanding for producing human insulin and functional protein. The procedure for αB-Cry-mediated insulin folding could be also applicable for the large-scale production of this highly important therapeutic peptide hormone.

Highlights

Changes in diet and lifestyle are two important factors causing the worldwide dramatic increase in the incidence of diabetes [1]
Dual role of human αB-crystallin in insulin production products in the presence of CNBr that would otherwise need to be removed from the final product (Fig 1)
The Asp-Pro sequence in αB-Cry is sensitive to the cleavage by formic acid and this reagent is needed for releasing of the insulin chains from the fusion proteins in the cleavage step

Summary

Introduction

Changes in diet and lifestyle are two important factors causing the worldwide dramatic increase in the incidence of diabetes [1]. Dual role of human αB-crystallin in insulin production from insulin-dependent diabetes mellitus [2]. Both type I and type II diabetic patients require insulin, but because of developing insulin resistance, the late stage type II diabetes patients use larger amounts of this hormone [3, 4]. Due to the cumulative incidence of this metabolic disease among children and adults, insulin is at the top of the list of therapeutic peptides in high demand [5]. Insulin controls the storage and use of sugars and if by its dysfunction, the blood sugar homeostasis is not well preserved for long periods, serious complications will appear in different tissues [6]

Methods

Results

Conclusion