• KSBB Journal
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• v.11 no.2
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• pp.165-172
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• 1996

To obtain a correctly folded human proinsulin, export of proinsulin using Staphylococcal protein A signal sequence-mediated secretion pathway has been attempted in E.coli. A secretion operon for proinsulin was constructed by consecutively connecting T7 promoter, SPA ribosome binding site, SPA signal sequence gene, and human proinsulin gene. Little immunoreactive proinsulin was detected in the periplasmic space and. culture medium, and not even in cytoplasmic space. The qualitative analysis of transcribed proinsulin mRNA and the in vitro transcription/translation experiment suggests that the negligible level of proinsulin export appears to be due to intracellular degradation of proinsulin, rather than due to the blockage during translocation. However, expression of proinsulin fusion protein such as MBP-proinsulin could dramatically increase export of proinsulin in E.coli.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.690-693
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• 2000

Efficient intron deletion with the correct splicing of the two exons of the human proinsulin gene was accomplished by a novel stepwise method using genomic DNA [5]. The two exons were separately amplified in two steps, using the second step primers that incorporated additional bases complementary to the other exon. The fragments were combined in a third PCR reaction. Cloning and sequencing of the PCR product demonstrated the correct splicing of the two exons. Expression studies, using the pET9a vector, revealed a protein band with the correct size with respect to human proinsulin as confirmed by SDS-PAGe and Western blot. Proinsulin concentration was estimated to be around 200 mg per liter culture, expressed as inclusion bodies. Protein secretion to the culture medium and periplasmic space was achieved by cloning in the pEZZ18 vector.

• 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.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.75-80
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• 2000

An improved method of refolding recombinant human proinsulin from E. coli was presented. It was based on a two-stage stirred tank reactor in which denatured proinsulin-s-sulfonate was mixed instantaneously with a reaction buffer in the first stage reactor, and then fed to the second stage reactor. The mixture was stirred further for a total of 30h in the second stage reactor. In this system, unfavorable effects present due to the increase in reaction volume and protein concentration for protein refolding, which becomes significant in a large-scale operation, were avoided. Refolding yields of over 80% was obtained for achieving reaction volume of upto 50 l at protein concentration of 1 mg/ml. The optimum urea concentration was 1M. Refolding yield at the 1-1 reaction volume and protein concentration of 0.5mg/ml was increased about 2.5-fold, compared to that in a batch reactor. By increasing protein concentration in a two-stage refolding reaction, the cost for insulin production could be reduced, therefore, making this process economical.

• 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.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.10a
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• pp.41-48
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• 1995

To increase the folding efficiency of proinsulin, we have designed a series of mini-proinsulins having the central C-peptide region replaced with sequences forming reverse turns. These proteins were produced as fusion proteins in E. coli in the form of inclusion bodies. After isolation process of the sulfonated mini-proinsulins, the subsequent refolding experiments indicate that the mini-proinsulins, with non-native penta-peptide sequences inserted between two of the enzyme processing sites, show substantially increased folding yields compared with the proinsulin. The correct disulfide connections were verified by fingerprint analysis using Glu-C endoproteinase. These novel mini-proinsulins could be used for the study of folding mechanism of proinsulin.

• BMB Reports
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• v.33 no.2
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• pp.120-125
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• 2000

M2PI is an active single chain mini-proinsulin with a 9-residue linker containing the turn-forming sequence 'YPGDV' between the B- and A-chains, but which retains about 50% of native insulin receptor binding activity. The refolding efficiency of M2PI is higher than proinsulin by 20-40% at alkaline pH, and native insulin is generated by the enzymatic conversion of M2PI. The solution structure of M2PI was determined by NMR spectroscopy. The global structure of M2PI is similar to that of native insulin, but the flexible linker between the B- and A-chains perturbed the N-terminal A-chain and C-terminal B-chain. The helix in the N-terminal A-chain is partly perturbed and the ${\beta}$-turn in the B-chain is disrupted in M2PI. However, the linker between the two chains was completely disordered indicating that the designed turn was not formed under the experimental conditions (20% acetic acid). Considering the fact that an insulin analogue, directly cross-linked between the C-terminus of the B-chain and the N-terminus of the A-chain, has negligible binding activity, a flexible linker between the two chains is sufficient to keep binding activity of M2PI, but the perturbed secondary structures are detrimental to receptor binding.

• 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.

• The Korean Journal of Nuclear Medicine Technology
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• v.17 no.1
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• pp.76-79
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• 2013

Purpose: It is very important to establish the appropriate reference range in the laboratory for preventing mistakes like false positive or false negative. Because the reference range in the laboratory is standard of patient test results interpretation. Proinsulin is precursor hormone of insulin, and the importance is increasing for diagnosing diabetes or insulinoma. Proinsulin reagent used in our laboratory is produced in the USA, and the reference range provided by manufacturer was adapted to our reference range after the validation test. But, it is generally recommend for the every laboratory to establish the their own reference range. So, we decided to re-evaluate the reference range with our patients' test results. Materials and Methods: Among 737 patients who had been to health promotion center in our hospital between Dec. $8^{th}$ 2011 and Dec. $21^{st}$ 2011, 563 patients are chosen with exception of diabetics patients and patients showing abnormal test results in Fasting Glucose, HbA1c, Insulin, and C-peptide. The 563 test results (275 males and 288 females) were classified with three groups(entire, male, female), and analysis of normal distribution was performed with aid of SPSS(version 19.0). Because Each group didn't show normal distribution, the reference range was set from the lowest limit of 2.5% to the highest limit of 97.5% with Percentile method used in non-normal distribution. Results: When evaluation values are sorted in ascending order, the entire range is 4.5~52.0 pM and 5.3~51.9 pM for male and 4.5~52.0 pM for female. The calculated reference range with percentile method shows 6.7~26.5 pM for entire group, 6.8~26.5 pM for male and 6.7~26.5 pM for female, respectively. Conclusion: The reference range provided by reagent manufacturer is 6.4~9.4 pM and the one established in this study is 6.7~26.5 pM. This difference might be caused by racial characteristics between Western people and Koreans. So an ideal reference range can be gotten with normal population visiting to every hospital. Our hospital has been using the newly re-establishing reference range under consultation with the department of endocrinology since Aug. $1^{st}$ 2012.

• Bulletin of the Korean Chemical Society
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• v.18 no.8
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• pp.798-800
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• 1997