• Journal of Microbiology and Biotechnology
• /
• 제18권5호
• /
• pp.983-989
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• 제26권10호
• /
• pp.1781-1789
• /
• 2016

Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide glargine insulin when transformed into Escherichia coli JM109. The transformed E. coli cells were cultured by fed-batch fermentation. The final dry cell mass was 18 g/l. The prepeptide glargine insulin was 38.52% of the total protein. It was expressed as an inclusion body and then refolded to recover the biological activity. To convert the prepeptide into glargine insulin, citraconylation and trypsin cleavage were performed. Using citraconylation, the yield of enzymatic conversion for glargine insulin increased by 3.2-fold compared with that without citraconylation. After the enzyme reaction, active glargine insulin was purified by two types of chromatography (ion-exchange chromatography and reverse-phase chromatography). We obtained recombinant human glargine insulin at 98.11% purity and verified that it is equal to the standard of human glargine insulin, based on High-performance liquid chromatography analysis and Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry. We thus established a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. This established process for recombinant human glargine insulin may be a model process for the production of other human insulin analogs.

• Journal of Plant Biotechnology
• /
• 제40권3호
• /
• pp.169-177
• /
• 2013

최근 급속도로 당뇨병 환자가 증가하면서 인슐린 시장이 크게 성장하고 있다. 또한 최근 식물체를 이용하여 의약용 단백질 생산이 경제적인 측면과 안정성 측면에서 매우 효과적임이 보고되고 있어 이를 이용한 분자농업이 주목을 받고 있다. 본 연구에서는 인슐린 단백질을 식물체에서 생산하기 위한 유전자 발현 construct를 설계하기 위한 실험으로서 식물발현용 preprominiinsulin construct를 제조하기 위한 단계적 실험을 수행하였다. 우선 proinsulin이 무세포 식물 전사/번역시스템에서 성공적으로 발현됨을 확인하였다. Prominiinsulin construct를 제조하여 대장균에서 발현시키는데 성공하였으며, 이를 트립신으로 절단한 후 인간 항인슐린 항체를 이용한 western 분석을 통하여 효과적으로 A-펩타이드와 B-펩타이드가 형성되며 이후 적절하게 접힘이 일어나고 hexamer로 조립됨을 확인하였다. 이후 식물체에서 재조합 인슐린 유전자가 발현되는지를 확인하기 위하여 RFP 결합 construct를 제조하여 담배의 현탁배양세포인 BY-2 세포에 형질전환시켜 RFP 결합 preprominiinsulin이 성공적으로 발현됨을 확인 하였다. 이러한 성공적인 연구 결과를 토대로 향후 이 construct는 RFP 단백질을 제거하여 35S 프로모터에 직접 유도되는 [N 말단 ${\rightarrow}$ tobacco E2 시그널 펩타이드 ${\rightarrow}$ B-펩타이드(1-29 AA) ${\rightarrow}$ AAK ${\rightarrow}$ A-펩타이드(1-21 AA) ${\rightarrow}$ RR ${\rightarrow}$ His6 ${\rightarrow}$ KDEL ${\rightarrow}$ C 말단] construct를 제조하여 담배 식물체에 형질전환시켜 분자농업을 통해 인간 인슐린 단백질을 생산하는데 활용하고자 한다.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XII)
• /
• pp.34-38
• /
• 2003

For the production of $B^{30}-homoserine$ insulin analog as a novel anti-diabetic drug, the fermentative study was attempted for the maximal gene expression of HTS-fused $B^{30}-homoserine$ insulin precursor in the recombinant Escherichia coli cells. In a batch fermentation, the maximal production of insulin precursor as much as 38.95 mg/L-h, which occupied more than 12.8% of total cell protein. was achieved when the gene expression was induced by 0.5 mM IPTG at the middle logarithmic growth phase. The HTS-fused $B^{30}-homoserine$ insulin precursor was recovered from a batch culture through the processes of cell harvest, collection of insoluble fraction after sonication and purification by nickel affinity column chromatography. The isolated insulin precursor was 14 mg/L with a recovery yield of 35.9% of expressed gene product. The insulin A and B chain mixture was recovered after the insulin precursor was subjected to CNBr cleavage and purified by nickel affinity column chromatography. The isolated insulin chains were then sulfitolyzed with sodium thiosulfat and sodium tetrathionate, and reconstituted to insulin analog with ${\beta}-mercaptoethanol$, followed by purification with CM-Sepharose C-25 column chromatography.

• Genomics & Informatics
• /
• 제15권4호
• /
• pp.142-146
• /
• 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.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제1권1호
• /
• pp.9-12
• /
• 1996

In order to produce the single chain precursor of a novel human insulin analogue, (B30-Homoserine) insulin, the fermentative behaviors of Escherichia coli JM103 were studied, which harbors pKBA plasmid carrying a hybrid gene in which the gene for a single chain precursor was fused with lacZ gene under tac promoter. The maximal induction of gene expression was achieved when more than 0.05 mM of isopropyl-$\beta$-D-thiogalactopyranoside(IPTG) was supplemented to fermentation medium after 4 h cultivation of E. coli, and followed by longer than 2-h fermentation. The hybrid protein of the single chain insulin precursor was isolated from cytoplasmic inclusion bodies by dissolving in 8M urea solution, and purified through DEAE-Sephacel and Sephadex G-200 column chromatographies with a recovery of 35%. The finally purified hybrid protein showed a single band on sodium dodecyl sulfate-polyacrylamide gel.

• Journal of Animal Science and Technology
• /
• 제50권2호
• /
• pp.177-184
• /
• 2008

Acid-labile subunit(ALS)는 85-kDa 크기의 당단백질로서 7.5-kDa의 insulin-like growth factor(IGF) 및 40~45-kDa IGF-binding protein-3와 결합하여 150-kDa ternary complex를 형성하는 혈장단백질이다. 선행연구에서 본 연구진은 reverse transcription-polymerase chain reaction(RT-PCR) 방법으로 돼지(porcine) ALS(pALS)의 coding sequence를 합성하여 plasmid vector에 삽입시켜 ‘expression construct’를 제작한 바 있다. 그러나 본 expression construct의 pALS coding sequence에는 PCR error로 추정되는 원인으로 말미암아 2개의 bases에서 mis-sense mutation이 일어난 것이 발견되었다. 본 연구에서는 ‘site-directed mutagenesis’ 방법으로 pALS의 올바른 coding sequence를 합성하여 ‘insert DNA’의 마지막 codon 다음에 ‘His-tag’ sequence가 위치한 pET- 28a(+) plasmid expression vector에 삽입하였다. 본 expression construct는 E. coli BL21(DE3) 세포에서 ‘induction’ 시켰고, 발현된 유전자재조합(recombinant) peptide는 Ni-affinity chromato- graphy로 정제하였다. 이렇게 affinity chro- matography로 정제된 peptide는 SDS-PAGE에서 66kDa 위치에 single band를 나타냄으로써 recombinant pALS의 예상된 질량과 일치하였다. 이상의 결과는 본 연구에서 recombinant pALS peptide가 성공적으로 발현정제되었음을 시사한다.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제6권2호
• /
• pp.144-149
• /
• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• The Korean Journal of Physiology and Pharmacology
• /
• 제17권2호
• /
• pp.157-162
• /
• 2013

Insulin-like growth factor binding proteins (IGFBPs) are important components of insulin growth factor (IGF) signaling pathways. One of the binding proteins, IGFBP-5, enhances the actions of IGF-1, which include the enhanced proliferation of smooth muscle cells. In the present study, we examined the expression and the biological effects of IGFBP-5 in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). The levels of IGFBP-5 mRNA and protein were found to be higher in the VSMC from SHR than in those from WKY. Treatment with recombinant IGFBP-5-stimulated VSMC proliferation in WKY to the levels observed in SHR. In the VSMCs of WKY, incubation with angiotensin (Ang) II or IGF-1 dose dependently increased IGFBP-5 protein levels. Transfection with IGFBP-5 siRNA reduced VSMC proliferation in SHR to the levels exhibited in WKY. In addition, recombinant IGFBP-5 significantly up-regulated ERK1/2 phosphorylation in the VSMCs of WKY as much as those of SHR. Concurrent treatment with the MEK1/2 inhibitors, PD98059 or U0126 completely inhibited recombinant IGFBP-5-induced VSMC proliferation in WKY, while concurrent treatment with the phosphatidylinositol-3 kinase inhibitor, LY294002, had no effect. Furthermore, knockdown with IGFBP-5 siRNA inhibited ERK1/2 phosphorylation in VSMC of SHR. These results suggest that IGFBP-5 plays a role in the regulation of VSMC proliferation via ERK1/2 MAPK signaling in hypertensive rats.

• Asian-Australasian Journal of Animal Sciences
• /
• 제17권8호
• /
• pp.1062-1068
• /
• 2004

Leptin has a major role in the regulation of food intake and energy homeostasis. In addition, leptin participates in many physiological functions including regulation of lipid metabolism. Bovine recombinant leptin protein was produced in E. coli cells in order to understand function of leptin in the regulation of lipid metabolism. The leptin expression vector was constructed in pGEX-4T-3 vector and transformed into E. coli BL21 cells. Expression of the GST-leptin fusion protein was induced with IPTG. The fusion protein was purified using glutathione sepharose 4B batch method, and the recombinant leptin was eluted after thrombin protease digestion. The effect of leptin on glucose transport was examined in the differentiated adipocytes of 3T3-L1 cells. Leptin had no effect on basal and insulin-stimulated glucose transport in 3T3-L1 cells (p>0.05). Effect of recombinant leptin on glucose and acetate transport was examined in adipose tissues of Korean cattle (Hanwoo). Insulin stimulated glucose transport in both intramuscular and subcutaneous adipose tissues (p<0.05), but leptin did not affect glucose transport in both adipose tissues (p>0.05). Insulin stimulated acetate transport in bovine adipose tissues (p<0.05), but leptin did not affect acetate transport (p>0.05). Northern and RT-PCR analyses showed that mRNA levels of uncoupling protein-2 were increased by leptin treatment in 3T3-L1 cells without statistical difference (p>0.05). In conclusion, bovine recombinant leptin did not affect glucose and acetate transport in both 3T3-L1 adipocytes and bovine adipose tissues, while it stimulates UCP-2 mRNA expression in 3T3-L1 cells.