대장균과 식물시스템에서 재조합 인간 prominiinsulin 생합성 분석
Biosynthesis of recombinant human prominiinsulin in E. coli and plant systems
2
조회
0
다운로드
0
인용
0
참고문헌
초록
Recently, the number of people with diabetes is rapidly increasing, coupled with the fact that the insulin market is remarkably increasing. Therefore, molecular farming for plant-derived pharmaceutical protein production is reported as becoming more attractive than ever. In this study, we carried out experiments step by step for development of recombinant insulin constructs, which were transformed into E. coli system, in vitro transcription and translation system, and tobacco cells. At first, recombinant proinsulin protein was successfully produced in in vitro transcription and translation system with wheat germ extract. After which, recombinant construct of prominiinsulin encoded a fusion protein of 7.8 kDa with trypsin cleavage sites at N terminus and C terminus of minimized C-peptide was tried to in vitro expression using E.coli culture. After purification with His-tag column, the resulting recombinant prominiinsulin protein was processed with trypsin, and then checked insulin biosynthesis by SDS-PAGE and western blot analysis with anti-insulin monoclonal antibody. The immunoreactive product of trypsin-treated miniinsulin was identical to the predicted insulin hexamer. The construct of 35S promoter-driven preprominiinsulin recombinant gene with signal peptide region for ER-targeting and red fluorescence protein gene [N terminus → tobacco E2 signal peptide → B-peptide (1-29 AA) → AAK → A-peptide (1-21 AA) → RR → His6 → KDEL → C terminus] was transformed into BY-2 tobacco cells. A polypeptide corresponding to the 38-kDa molecular mass predicted for fusion protein was detected in total protein profiles from transgenic BY-2 cells by western analysis. Therefore, this recombinant preprominiinsulin construct can be used for generation of transgenic tobacco plants producing therapeutic recombinant insulin.
Abstract
Recently, the number of people with diabetes is rapidly increasing, coupled with the fact that the insulin market is remarkably increasing. Therefore, molecular farming for plant-derived pharmaceutical protein production is reported as becoming more attractive than ever. In this study, we carried out experiments step by step for development of recombinant insulin constructs, which were transformed into E. coli system, in vitro transcription and translation system, and tobacco cells. At first, recombinant proinsulin protein was successfully produced in in vitro transcription and translation system with wheat germ extract. After which, recombinant construct of prominiinsulin encoded a fusion protein of 7.8 kDa with trypsin cleavage sites at N terminus and C terminus of minimized C-peptide was tried to in vitro expression using E.coli culture. After purification with His-tag column, the resulting recombinant prominiinsulin protein was processed with trypsin, and then checked insulin biosynthesis by SDS-PAGE and western blot analysis with anti-insulin monoclonal antibody. The immunoreactive product of trypsin-treated miniinsulin was identical to the predicted insulin hexamer. The construct of 35S promoter-driven preprominiinsulin recombinant gene with signal peptide region for ER-targeting and red fluorescence protein gene [N terminus → tobacco E2 signal peptide → B-peptide (1-29 AA) → AAK → A-peptide (1-21 AA) → RR → His6 → KDEL → C terminus] was transformed into BY-2 tobacco cells. A polypeptide corresponding to the 38-kDa molecular mass predicted for fusion protein was detected in total protein profiles from transgenic BY-2 cells by western analysis. Therefore, this recombinant preprominiinsulin construct can be used for generation of transgenic tobacco plants producing therapeutic recombinant insulin.
참고문헌 (20)
- Cardinale F, "Convergence and Divergence of Stress-Induced Mitogen-Activated Protein Kinase Signaling Pathways at the Level of Two Distinct Mitogen-Activated Protein Kinase Kinases", Plant Cell, 14, pp.703~711 (2002)
- Cornelissen BJ, "Bol JF(1986)A tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatin", Nature, 321, pp.531~532 (1986)
- Cowley DJ, "Expression, purification and characterization of recombinant human proinsulin", FEBS Lett, 402, pp.124~130 (1997)
- Dunn MF, "Zinc-ligand interactions modulate assembly and stability of the insulin hexamer - a review", Biometals, 18, pp.295~303 (2005)
- Farinas CS, "Aqueous extraction of recombinant human proinsulin from transgenic maize endosperm", Biotechnol Prog, 21, pp.1466~1471 (2005)
- Hu X, "Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein", Plant Mol Biol, 34, pp.949~959 (1997)
- 김미경, "소포체 스트레스(Endoplasmic Reticulum Stress)와 당뇨병", Endocrinology and Metabolism, 23(1), pp.1~8 (2008), 대한내분비학회
- Liu M, "Proinsulin misfolding and diabetes: mutant INS gene-induced diabetes of youth", Trends Endocrinol Metab, 21, pp.652~659 (2010)
- Nykiforuk CL, "Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds", Plant Biotechnol J, 4, pp.77~85 (2006)
- Olsen JV, "Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues", Mol Cell Proteomics, 3, pp.608~614 (2004)
- Pietropaolo M, "Persistent C-peptide: what does it mean?", Curr Opin Endocrinol Diabetes Obes, 20, pp.279~284 (2013)
- Puigbò P, "E-CAI: a novel server to estimate an expected value of Codon Adaptation Index (eCAI)", BMC Bioinformatics, 9, pp.1471~2105 (2008)
- Qiao ZS, "In vitro refolding of human proinsulin. Kinetic intermediates, putative disulfide-forming pathway folding initiation site, and potential role of C-peptide in folding process", J Biol Chem, 278, pp.17800~17809 (2003)
- Sambrook J, "Molecular cloning: A Laboratory Mannual. Cold Spring Harbor Laboratory Press" (2001), Cold Spring Harbor
- van Kan JAL, "Structure of tobacco genes encoding thaumatin-like proteins", Plant Mol Biol, 12, pp.153~155 (1989)
- van Loon LC, "Identification, purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves", Plant Mol Biol, 9, pp.593~609 (1987)
- Wahren J, "C-peptide makes a comeback", Diabetes Metab Res Rev, 19, pp.345~347 (2003)
- Weiss MA, "Diabetes mellitus due to the toxic misfolding of proinsulin variants", FEBS Lett, 587, pp.1942~1950 (2013)
- Zamani A, "Gene Cloning and Tissue Expression Analysis of a PR-5 Thaumatin-Like Protein in Phellinus weirii-Infected Douglas- Fir", Phytopathology, 94, pp.1235~1243 (2004)
- "WO 10-2006-0052705 (2006) Methods for the production of insulin in plants. SemBioSys Genetics Incorporated", ~ (2006)