title: High-level expression and efficient one-step chromatographic purification of a soluble human leukemia inhibitory factor (LIF) in Escherichia coli.
abstract: Leukemia inhibitor factor (LIF) is a three disulfide bridge-containing cytokine with numerous regulatory effects. In this report, we present the high level expression of a soluble recombinant human LIF (rhLIF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hLIF cDNA was cloned into pET3b vector, and transformed into E. coli OrigamiB(DE3) harboring the bacterial thioredoxin coexpression vector. By using an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™), the yield of soluble proteins was significantly improved in comparison to commonly-used 2 × YT media. The recombinant protein was purified via a single chromatographic step using an affinity tag-based protein purification system that processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Soluble rhLIF yield was estimated to be approximately 1mg/g of wet weight cells, with above 98% purity. The rhLIF protein specifically inhibited the proliferation of the murine myeloblastic leukemia M1 cell in a dose-dependent manner, and induced Stat3 phosphorylation in mouse ES cells. This novel expression and purification protocol for the production of recombinant hLIF is a simple, suitable, and effective method.
authors: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors: Keitaro
authors: K
authors: Imaizumi
authors: Shin-Ichi
authors: S
authors: Nishikawa
authors: Hiroshi
authors: H
authors: Tarui
authors: Teruo
authors: T
authors: Akuta
mesh_terms: D000818:Animals; D053595:Embryonic Stem Cells / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse