title: Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli.
abstract: Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E. coli because the proteins of extremophiles are strongly resistant to extreme conditions. In a previous study, a thermostable esterase Est-AF was isolated from Archaeoglobus fulgidus and expressed in E. coli. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 ± 0.35 mg/L to 61.75 ± 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-d-1-thiogalactoside (IPTG) at 37 °C for 5 h. Under these conditions, the expression level of Est-AF was increased from 31.5 ± 0.35 mg/L to 119.52 ± 0.34 mg/L.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, 16499, South Korea.
authors.forename: Jinyeong
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, 16499, South Korea.
authors.forename: Seul I
authors.initials: SI
authors.lastname: Kim
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, 16499, South Korea.
authors.forename: Eunsoo
authors.initials: E
authors.lastname: Hong
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, 16499, South Korea. Electronic address: ywryu@ajou.ac.kr.
authors.forename: Yeonwoo
authors.initials: Y
authors.lastname: Ryu
mesh_terms: D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019611:Archaeoglobus fulgidus / Q000201:enzymology / Q000235:genetics*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D004950:Esterases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D006358:Hot Temperature; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D004950:Esterases
keywords: Archaeoglobus fulgidus; Escherichia coli; Extremophile; Heterologous protein expression; Thermostable esterase