• KSBB Journal
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• 제11권2호
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• pp.165-172
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• 1996

자신의 3차구조를 가진 인체 프로인슐린을 얻기 위하여 Staphylococcal 프로테인 A(SPA)의 신호 랩타이드를 이용하여 대장균내에서 분비 발현을 시도하였다. 분비 발현을 위해 T7 프로모터, SPA 리보좀 바인팅 부위, SPA 신호 랩타이드, 프로인슐린 유전자를 연속적으로 연결하여 분비 벡터를 구성하였다. 이 벡터를 대장균에 넣은후 발현을 유도했으 나 면역적으로 반응하는 인체 프로인슐린은 배양액이나 페리프라스믹 공간에서 거의 존재하지 않았으며 세포 내에도 존재하지 않았다. 그러나 말현 유도시 세포 내에 프로인슐린 RNA가 급격히 증가하였으며 구성한 벡터는 실험실적으로 프로인슐린을 전 사(transcription) , 번역 (translation) 할 수 있었다. 이는 프로인슐린이 번역 후 급히 세포내에서 분 해됨을 의미하며 이로 인해 분비된 프로인슐린을 거의 얻을 수 없게 된 것으로 생각된다. 그러나 프로인 슐린의 세포내 안정성을 위해 말토즈 바인딩 프로테인을 융합짝으로 프로인슐린에 연결한 경우 과량의 분비된 인체 프로인슐린을 검출할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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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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• 제15권4호
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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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• 제10권1호
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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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• 제41권8호
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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.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 제3회 추계심포지움
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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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• 제33권2호
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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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• 제25권10호
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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.

• 핵의학기술
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• 제17권1호
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• pp.76-79
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• 2013

검사실에서 사용하는 참고치는 검사결과의 해석에 기준이 되는 것으로 적절치 못한 경우 위양성이나 위음성과 같은 오류를 범할 수 있어 올바른 참고치 설정은 매우 중요하다. Proinsulin은 insulin의 전구체물질로서 당뇨병과 인슐린종의 판단에 중요한 검사로 사용되고 있다. 현재 본원 검사실에서 사용되는 Proinsulin 시약은 미국에서 제조되었고 참고치 또한 제조사에서 제공하는 수치를 검증을 통해 사용하였다. 그러나 통상적인 추천은 각 검사실에서 검사실 집단에 적합한 참고치를 도출하여 사용하는 것을 권고하고 있다. 따라서 본원에 내원하는 수진자를 대상으로 참고치를 재평가하기로 하였다. 2011년 12월 8일부터 21일까지 본원 건강의학센터에 내원한 수진자 737명 중 당뇨병 진단을 받은 환자와 Fasting Glucose, HbA1c, Insulin, C-peptide 검사에서 참고치를 벗어 난 환자를 제외한 563명을 대상으로 하였다. 대상자 563명 (남자 275명, 여자 288명)을 3가지 집단(전체, 남자, 여자)으로 구분하고, SPSS(version 19.0)를 사용하여 정규분포 검증을 실시하였다. 각각의 집단 모두 정규분포를 이루지 않아 비정규분포 시 사용하는 Percentile법으로 하한 2.5%에서 상한 97.5%를 참고치 범위로 설정하였다. 전체 대상의 측정값을 크기 순으로 나열하면 4.5~52.0 pM의 범위로 남자 5.3~51.9 pM, 여자 4.5~52.0 pM의 범위를 보였다. Percentile 법으로 하한 2.5%에서 상한 97.5%로 설정한 경우에는 전체535명(남자 259명, 여자 276명)으로 6.7~26.5 pM의 범위였으며 남자 6.8~26.5 pM, 여자 6.7~26.5 pM의 범위를 보였다.Proinsulin 시약 제조사에서 제공된 참고치는 6.4~9.4 pM이며 본 연구에서 재설정된 참고치는 6.7~26.5 pM이다. 이 차이는 서양인과 한국인의 인종간 특이성 때문인 것으로 추정된다. 따라서 가장 이상적인 참고치는 각자의 병원에 내원하는 정상인들을 대상으로 설정하는 것이다. 본원은 내분비내과와 협의를 통해 2012년 8월 1일부터 재설정된 참고치를 사용하고 있다.

• Bulletin of the Korean Chemical Society
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• 제18권8호
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• pp.798-800
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• 1997