title: Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification.
abstract: Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.
authors.forename: Jung-A
authors.initials: JA
authors.lastname: Song
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: A Song
authors.initials: AS
authors.lastname: Jung
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: Bon-Kyung
authors.initials: BK
authors.lastname: Koo
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: Seon-Ha
authors.initials: SH
authors.lastname: Chong
authors.affiliation: Graduate School of Medical Science and Engineering Korea Advanced Institute of Science and Technology, Daejeon, South Korea.
authors.forename: Kyunhoo
authors.initials: K
authors.lastname: Kim
authors.affiliation: Graduate School of Medical Science and Engineering Korea Advanced Institute of Science and Technology, Daejeon, South Korea.
authors.forename: Dong Kyu
authors.initials: DK
authors.lastname: Choi
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: Thu Trang
authors.initials: TT
authors.lastname: Thi Vu
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: Minh Tan
authors.initials: MT
authors.lastname: Nguyen
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: Boram
authors.initials: B
authors.lastname: Jeong
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: Han-Bong
authors.initials: HB
authors.lastname: Ryu
authors.affiliation: Graduate School of Medical Science and Engineering Korea Advanced Institute of Science and Technology, Daejeon, South Korea.
authors.forename: Injune
authors.initials: I
authors.lastname: Kim
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: Yeon Jin
authors.initials: YJ
authors.lastname: Jang
authors.affiliation: Institute of Molecular and Cell Biology, ASTAR (Agency for Science, Technology and Research), Biopolis, Singapore, Singapore ; Department of Biochemistry, National University of Singapore, Singapore, Singapore.
authors.forename: Robert Charles
authors.initials: RC
authors.lastname: Robinson
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: Han
authors.initials: H
authors.lastname: Choe
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000235:genetics* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D019704:Protein Disulfide-Isomerases / Q000235:genetics* / Q000378:metabolism; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D053605:Leukemia Inhibitory Factor; D011993:Recombinant Fusion Proteins; D019704:Protein Disulfide-Isomerases