• Applied Biological Chemistry
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• v.41 no.8
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• pp.572-577
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• 1998

The solution conformation of the human insulin precursor, preproinsulin, is described in terms of NMR spectral data. NMR experiments were performed on preproinsulin, whose structure was compared with the NMR structure of native human insulin. Despite the presence of the C-peptide and/or the signal peptide, secondary structure analyses indicate that the native structures of the A and B chains are well conserved even in preproinsulin. The observed relative robustness of the native structure in precursor forms permits further protein engineering experiments where the C-peptide or N-terminal signal sequence can be altered for the purpose of increasing expression or purification yields when producing recombinant human insulin.

• Applied Biological Chemistry
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• v.41 no.8
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• pp.568-571
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• 1998

The conformations of human insulin precursors, proinsulin and preproinsulin, are described in terms of molecular dynamics simulations. Despite the presence of the C-peptide and/or the signal peptide, molecular dynamics calculations utilizing the hydration shell model over a period of 500 ps indicate that the native conformations of the A and B chains are well conserved in both cases. These results further support the NMR spectroscopy results that the C-peptide is relatively disordered and does not influence the overall conformation of the native structure. The robustness of the native structure as demonstrated by experiment and simulation will permit future protein engineering applications, whereby the expression or purification yields can be improved upon sequence modification of the C-peptide and/or the signal peptide.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1742-1750
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• 2015

Human insulin is composed of 21 amino acids of an A-chain and 30 amino acids of a B-chain. This is the protein hormone that has the role of blood sugar control. When the recombinant human proinsulin is expressed in Escherichia coli, a serious problem is the formation of an inclusion body. Therefore, the inclusion body must be denatured and refolded under chaotropic agents and suitable reductants. In this study, H27R-proinsulin was refolded from the denatured form with β-mercaptoethanol and urea. The refolding reaction was completed after 15 h at $15^{\circ}C$, whereas the reaction at $25^{\circ}C$ was faster than that at $15^{\circ}C$. The refolding yield at $15^{\circ}C$ was 17% higher than that at $25^{\circ}C$. The refolding reaction could be carried out at a high protein concentration (2 g/l) using direct refolding without sulfonation. The most economical and optimal refolding condition for human preproinsulin was 1.5 g/l protein, 10 mM glycine buffer containing 0.6 M urea, pH 10.6, and 0.3 mM β-mercaptoethanol at $15^{\circ}C$ for 16 h. The maximum refolding yield was 74.8% at $15^{\circ}C$ with 1.5 g/l protein. Moreover, the refolded preproinsulin could be converted into normal mature insulin with two enzymes. The average amount of human insulin was 138.2 g from 200 L of fermentation broth after enzyme reaction with H27R-proinsulin. The direct refolding process for H27R-proinsulin was successfully set up without sulfonation. The step yields for refolding and enzyme reaction were comparatively high. Therefore, our refolding process for production of recombinant insulin may be beneficial to the large-scale production of other biologically active proteins.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.983-989
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• 2008

Human insulin is a hormone well-known to regulate the blood glucose level. Recombinant preproinsulin, a precursor of authentic insulin, is typically produced in E. coli as an inactive inclusion body, the solubilization of which needs the addition of reducing agents such as $\beta$-mercaptoethanol. To make authentic insulin, recombinant preproinsulin is modified enzymatically by trypsin and carboxypeptidase B. The effects of $\beta$-mercaptoethanol on the formation of human insulin derivatives were investigated in the enzymatic modification by using commercially available human proinsulin as a substrate. Addition of 1 mM $\beta$-mercaptoethanol induced the formation of various insulin derivatives. Among them, the second major one, impurity 3, was found to be identical to the insulin B chain fragment from $Phe_1$ to $Glu_{21}$. Minimization of the formation of insulin derivatives and concomitant improvement of the production yield of human insulin were achieved by the addition of hydrogen peroxide. Hydrogen peroxide bound with $\beta$-mercaptoethanol and thereby reduced the negative effects of $\beta$-mercaptoethanol considerably. Elimination of the impurity 3 and other derivatives by the addition of over 10 mM hydrogen peroxide in the presence of $\beta$-mercaptoethanolled to a 1.3-fold increase in the recovery efficiency of insulin, compared with those for the case without hydrogen peroxide. The positive effects of hydrogen peroxide were also confirmed with recombinant human preproinsulin expressed in recombinant E. coli as an inclusion body.

• Proceedings of the Korean Magnetic Resonance Society Conference
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• 1999.02a
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• pp.11-11
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• 1999

• Genomics & Informatics
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• v.15 no.4
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• pp.142-146
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• 2017

More effective production of human insulin is important, because insulin is the main medication that is used to treat multiple types of diabetes and because many people are suffering from diabetes. The current system of insulin production is based on recombinant DNA technology, and the expression vector is composed of a preproinsulin sequence that is a fused form of an artificial leader peptide and the native proinsulin. It has been reported that the sequence of the leader peptide affects the production of insulin. To analyze how the leader peptide affects the maturation of insulin structurally, we adapted several in silico simulations using 13 artificial proinsulin sequences. Three-dimensional structures of models were predicted and compared. Although their sequences had few differences, the predicted structures were somewhat different. The structures were refined by molecular dynamics simulation, and the energy of each model was estimated. Then, protein-protein docking between the models and trypsin was carried out to compare how efficiently the protease could access the cleavage sites of the proinsulin models. The results showed some concordance with experimental results that have been reported; so, we expect our analysis will be used to predict the optimized sequence of artificial proinsulin for more effective production.