The use of fusion proteins to express the active proteins in E. coli provides a platform for the study of structural proteomics and the production of therapeutic proteins. Several proteins such as maltose binding protein (MBP), thioredoxin and tRNA synthetase are known to be useful fusion partners, ...
The use of fusion proteins to express the active proteins in E. coli provides a platform for the study of structural proteomics and the production of therapeutic proteins. Several proteins such as maltose binding protein (MBP), thioredoxin and tRNA synthetase are known to be useful fusion partners, but the exact mechanism by which they enhance the solubility of target proteins is not well understood. In this study, we investigated the folding behavior of an efficient fusion partner, E. coli lysine tRNA synthetase (LysRS). This protein contains an N-terminal RNA-binding domain (LysN) and a C-terminal catalytic domain (LysC). Fusion with LysN alone enhances the solubility similar to or slightly less than LysRS. We have expressed LysRS, LysN and LysC, respectively, in E. coli as a soluble form. The proteins were purified by Ni2+-charged affinity chromatography. Near- and far-ultraviolet circular dichroism (CD) spectroscopy showed typical CD spectra with a prevalence of alpha-helices in these proteins, indicating that each domain exists in a native form. The melting temperatures (Tm) of LysRS was around 43°C, LysN and LysC were 35°C and 47°C, respectively, indicating that LysN and LysC do not interact significantly. Protein folding was analyzed by fluorescence measurements using a stopped-flow apparatus. The folding rate of LysN was 456±2/sec with single exponential mode at pH 7.5, room temperature. However, the folding rates of LysRS (366±1/sec) and LysC (204±8/sec) were lower than that of LysN, indicating that the N-terminal domain folds earlier than the C-terminal domain. The in vitro refolding analysis of the LysRS fusion partner with the N-terminal domain folding first, may provide a structural basis for the solubility enhancing effect of the N-terminal fusion partners on their C-terminally linked proteins.This study is to develop a process of effective insulin production in that it improves two major obstacles which proinsulin-utilizing process bears. One is to reduce the cost of process using chemicals such as CNBr, GuanidineHCl, Sodium sulphite, and Sodium tetrathionatn which contains over 50 percent of total cost and the other is to simplify the production process by using protein engineering to enhance folding efficiency.To increase the folding efficiency of insulin precursor and the production yield of insulin, we have designed mini-proinsulins (T2RM2PI, T2RM5PI, T2RM6PI), having the central C-peptide region replaced with a sequence forming β-turn. The mini-proinsulin was fused at the N-terminus to a 22-residue fusion partner containing His10 tag. The genes for the fusion proteins were inserted downstream of the T7 promoter of the expression plasmid pT7-7, and the fusion proteins were produced as inclusion bodies in the Escherichia coli cytoplasm at levels up to 35% of the total cell protein.The proteins were sulfonated and the mini-proinsulin were purified using ion-exchange chromatography. The refolding yields of T2RM2PI, T2RM5PI, and T2RM6PI were 20~40% better than that of proinsulin studied at the same molar concentrations, indicating that the short turn-forming sequence is more effective in the refolding process than the much longer C-peptide. Native human insulin was succesfully generated by subsequent enzymatic conversion of mini-proinsulin. The high expression level, enhanced refolding yield and successful generation of native human insulin makes this approach a favourable alternative to the currently used proinsulin process. Therefore, the high yield of refolding displayed by mini-proinsulins, T2RM2PI, T2RM5PI and T2RM6PI suggesting that this single-chained mini-proinsulin may provide a foundation in understanding production and structure of insulin in the active form as well as the role of C-peptide in the folding and activity of proinsulin.
The use of fusion proteins to express the active proteins in E. coli provides a platform for the study of structural proteomics and the production of therapeutic proteins. Several proteins such as maltose binding protein (MBP), thioredoxin and tRNA synthetase are known to be useful fusion partners, but the exact mechanism by which they enhance the solubility of target proteins is not well understood. In this study, we investigated the folding behavior of an efficient fusion partner, E. coli lysine tRNA synthetase (LysRS). This protein contains an N-terminal RNA-binding domain (LysN) and a C-terminal catalytic domain (LysC). Fusion with LysN alone enhances the solubility similar to or slightly less than LysRS. We have expressed LysRS, LysN and LysC, respectively, in E. coli as a soluble form. The proteins were purified by Ni2+-charged affinity chromatography. Near- and far-ultraviolet circular dichroism (CD) spectroscopy showed typical CD spectra with a prevalence of alpha-helices in these proteins, indicating that each domain exists in a native form. The melting temperatures (Tm) of LysRS was around 43°C, LysN and LysC were 35°C and 47°C, respectively, indicating that LysN and LysC do not interact significantly. Protein folding was analyzed by fluorescence measurements using a stopped-flow apparatus. The folding rate of LysN was 456±2/sec with single exponential mode at pH 7.5, room temperature. However, the folding rates of LysRS (366±1/sec) and LysC (204±8/sec) were lower than that of LysN, indicating that the N-terminal domain folds earlier than the C-terminal domain. The in vitro refolding analysis of the LysRS fusion partner with the N-terminal domain folding first, may provide a structural basis for the solubility enhancing effect of the N-terminal fusion partners on their C-terminally linked proteins.This study is to develop a process of effective insulin production in that it improves two major obstacles which proinsulin-utilizing process bears. One is to reduce the cost of process using chemicals such as CNBr, GuanidineHCl, Sodium sulphite, and Sodium tetrathionatn which contains over 50 percent of total cost and the other is to simplify the production process by using protein engineering to enhance folding efficiency.To increase the folding efficiency of insulin precursor and the production yield of insulin, we have designed mini-proinsulins (T2RM2PI, T2RM5PI, T2RM6PI), having the central C-peptide region replaced with a sequence forming β-turn. The mini-proinsulin was fused at the N-terminus to a 22-residue fusion partner containing His10 tag. The genes for the fusion proteins were inserted downstream of the T7 promoter of the expression plasmid pT7-7, and the fusion proteins were produced as inclusion bodies in the Escherichia coli cytoplasm at levels up to 35% of the total cell protein.The proteins were sulfonated and the mini-proinsulin were purified using ion-exchange chromatography. The refolding yields of T2RM2PI, T2RM5PI, and T2RM6PI were 20~40% better than that of proinsulin studied at the same molar concentrations, indicating that the short turn-forming sequence is more effective in the refolding process than the much longer C-peptide. Native human insulin was succesfully generated by subsequent enzymatic conversion of mini-proinsulin. The high expression level, enhanced refolding yield and successful generation of native human insulin makes this approach a favourable alternative to the currently used proinsulin process. Therefore, the high yield of refolding displayed by mini-proinsulins, T2RM2PI, T2RM5PI and T2RM6PI suggesting that this single-chained mini-proinsulin may provide a foundation in understanding production and structure of insulin in the active form as well as the role of C-peptide in the folding and activity of proinsulin.
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#융합짝 폴딩 효율성 미니-프로인슐린 베타 회전 술폰화 리폴딩 수율 fusion partner E. coli lysine tRNA synthetase solubility enhancing effect CD in vitro refolding
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