title: A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli.
abstract: Mouse embryonic stem cells (mESCs) rely on a cytokine named leukemia inhibitory factor (LIF) to maintain their undifferentiated state and pluripotency. However, the progress of mESC research is restricted and limited to highly funded laboratories due to the cost of commercial LIF. Here we presented the homemade hLIF which is biologically active. The hLIF cDNA was cloned into two different vectors in order to produce N-terminal His₆-tag and Trx-His₆-tag hLIF fusion proteins in Origami(DE3) Escherichia coli. The His₆-hLIF fusion protein was not as soluble as the Trx-His₆-hLIF fusion protein. One-step immobilized metal affinity chromatography (IMAC) was done to recover high purity (> 95% pure) His₆-hLIF and Trx-His₆-hLIF fusion proteins with the yields of 100 and 200 mg/l of cell culture, respectively. The hLIF fusion proteins were identified by Western blot and verified by mass spectrometry (LC/MS/MS). The hLIF fusion proteins specifically promote the proliferation of TF-1 cells in a dose-dependent manner. They also demonstrate the potency to retain the morphology of undifferentiated mESCs, in that they were positive for mESC markers (Oct-4, Sox-2, Nanog, SSEA-1 and alkaline phosphatase activity). These results demonstrated that the N-terminal fusion tags of the His₆-hLIF and Trx-His₆-hLIF fusion proteins do not interfere with their biological activity. This expression and purification approach to produce recombinant hLIF is a simple, reliable, cost effective and user-friendly method.
authors.affiliation: Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Ave., Nakhon Ratchasima 30000, Thailand.
authors.forename: Sumeth
authors.initials: S
authors.lastname: Imsoonthornruksa
authors.forename: Parinya
authors.initials: P
authors.lastname: Noisa
authors.forename: Rangsun
authors.initials: R
authors.lastname: Parnpai
authors.forename: Mariena
authors.initials: M
authors.lastname: Ketudat-Cairns
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D015398:Signal Transduction / Q000235:genetics
chemical_list: D053605:Leukemia Inhibitory Factor; D011993:Recombinant Fusion Proteins