title: Expression, purification and characterization of active untagged recombinant human leukemia inhibitory factor from E.coli.
abstract: Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is considered to be a pleiotropic cytokine and functions in both cell proliferation and differentiation. It is widely used in the culture of mouse embryonic stem cells and is implicated in the implantation of mouse model and possibly in humans. Great efforts have been made on the efficient generation of LIF to meet the requirement of this cytokine in biomedical research. However, because of the low expression level in the eukaryotic system and poor purification yields, recombinant human LIF has usually been expressed either as inclusion body or as fusion protein in E. coli (Escherichia coli). Here we introduce a simple method to express hLIF in a soluble form in E. coli and a subsequent purification method. The expression of hLIF was induced at a low temperature (16 °C) and most of the expressed hLIF was observed to be in a soluble form. Then by using three steps of chromatography, which could be easily scaled-up for industrial purposes, active untagged hLIF was purified with similar bioactivity compared to that of the commercial product. The endotoxin level of purified hLIF protein in our method was determined to be lower than 1EU/μg, which was also comparable to the commercial products. Furthermore, as hLIF was expressed in a soluble form, there was no need to develop the denaturation and renaturation methods. The yield of hLIF protein was evaluated to be approximately 0.7 mg hLIF from 1 g wet weight of E.coli in our method.
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China.
authors.forename: Xueyan
authors.initials: X
authors.lastname: Xi
authors.affiliation: Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China.
authors.forename: Xiaolu
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Guan
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Jin
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Song
authors.affiliation: Department of Medical Biology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China. Electronic address: du.boyu@hotmail.com.
authors.forename: Boyu
authors.initials: B
authors.lastname: Du
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D011994:Recombinant Proteins
keywords: Expression; Leukemia inhibitory factor; Purification