Human αB-crystallin as fusion protein and molecular chaperone increases the expression and folding efficiency of recombinant insulin
Mohsen Akbarian, Reza Yousefi
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 coliand 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.
Circular dichroism, Secondary structure, Biochemistry