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

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.

Inhibitory effect of coumarin and its analogs on insulin fibrillation /cytotoxicity is depend on oligomerization states of the protein

“…Any alteration in the tertiary structure of the protein can affect the intensity of the emission light. 4,33 To doing so, insulin (2 mg mL À1 ) was dissolved in the dened acid condition containing the indicated different concentrations of the analogs and aer the considered incubation time, the sample was diluted to 1.0 mg mL À1 of the protein.…”

“…2,3 Also, protein misfolding, amorphous and morphous aggregations of these molecules are known as a big challenge in the industrial production of therapeutic proteins and peptides. 1,4 Therefore, the stabilization of these biopharmaceutical products in different ways is an important eld in the world of biotechnology. Several studies have been carried out to increase the stability of therapeutic proteins.…”

Characterization of insulin cross-seeding: the underlying mechanism reveals seeding and denaturant-induced insulin fibrillation proceeds through structurally similar intermediates

“…In the current study, the chain combination method was used for producing recombinant human insulin. 21 In brief, the plasmids (pET28b+) containing aB-BC (aB-Cry + B-chain) and aB-AC (aB-Cry + A-chain) fusion genes were constructed. Isopropyl b-D-1-thiogalactopyranoside (IPTG)-mediated expression of the fusion genes were done in Escherichia coli BL21 (DE3) pLysS cells (Invitrogen, Carlsbad, CA).…”

“…Aer combination of these two chains (see ref. 21), natively folded insulin was obtained using phenyl Sepharose hydrophobic column (GE Healthcare, Chicago IL). To produce a mutant form of aB-Cry, Quick-change site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used.…”

“…The concentrations of recombinant human insulin, insulin Achain, insulin B-chain and the fusion proteins (aB-AC and aB-BC) were estimated based on previous publications. 6,21,25 Also, following previous literature, 27 aA-Cry concentration was determined by UV absorption at 280 nm (an extinction coefficient of 0.72, mL mg À1 cm À1 , for 1.0 mg mL À1 ).…”