Prokaryotic production of recombinant proteins by fusion technology and genetically engineered E. coli strains : 원핵 생물에서의 융합 기술과 유전자 공학으로생성된 대장균에서의 재조합 단백질의 생산원문보기
NGUYENMINHTAN
(the Graduated School of the University of Ulsan
의과학전공
국내박사)
Escherichia coli (E. coli) is one of the most common expression systems used for production of recombinant human proteins. However, attempts to express recombinant human proteins in E. coli result in protein misfolding and aggregation into inclusion bodies, hampering purification. In concordance wit...
Escherichia coli (E. coli) is one of the most common expression systems used for production of recombinant human proteins. However, attempts to express recombinant human proteins in E. coli result in protein misfolding and aggregation into inclusion bodies, hampering purification. In concordance with other studies on cytoplasmic expression of human recombinant proteins, the E. coli-expressed human vascular endothelial growth factor (VEGF), human Oncostatin M (OSM) and human serum albumin (HSA) tend to be misfolded and forms inclusion bodies, resulting in poor solubility. Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a synthetic peptide derived from a random hexapeptide library. Due to its short half-life, the peptide was combined with Alb1 Nanobody as a fusion platform with the aim is to investigate whether enhanced in vivo half-life of the peptide would improve its therapeutic efficacy. In this study, a series of N-terminal fusion variants of the four proteins with hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a') were created to compare and characterize the soluble expression of the proteins in different E. coli host strains. In addition, the results propose that the utility of fusion platforms and genetically modified E. coli strains could be capable to offer the successful production of not only the four proteins but also other difficult-to-express and disulfide-rich proteins. Thus far, this is the first report on successfully expressing and purifying three human recombinant proteins, VEGF, OSM, rHSA and a half-life extended WKYMVM as soluble form in E. coli.
Escherichia coli (E. coli) is one of the most common expression systems used for production of recombinant human proteins. However, attempts to express recombinant human proteins in E. coli result in protein misfolding and aggregation into inclusion bodies, hampering purification. In concordance with other studies on cytoplasmic expression of human recombinant proteins, the E. coli-expressed human vascular endothelial growth factor (VEGF), human Oncostatin M (OSM) and human serum albumin (HSA) tend to be misfolded and forms inclusion bodies, resulting in poor solubility. Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a synthetic peptide derived from a random hexapeptide library. Due to its short half-life, the peptide was combined with Alb1 Nanobody as a fusion platform with the aim is to investigate whether enhanced in vivo half-life of the peptide would improve its therapeutic efficacy. In this study, a series of N-terminal fusion variants of the four proteins with hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a') were created to compare and characterize the soluble expression of the proteins in different E. coli host strains. In addition, the results propose that the utility of fusion platforms and genetically modified E. coli strains could be capable to offer the successful production of not only the four proteins but also other difficult-to-express and disulfide-rich proteins. Thus far, this is the first report on successfully expressing and purifying three human recombinant proteins, VEGF, OSM, rHSA and a half-life extended WKYMVM as soluble form in E. coli.
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