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A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
23890 43481 2011 8 PDF Available
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.

Keywords
hLIF, human leukemia inhibitory factor; Trx-tag, thioredoxin tag; His6-tag, hexahistidine-tag; IMAC, immobilized metal affinity chromatography; mESCs, mouse embryonic stem cells; MEF, mouse embryonic fibroblast; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe
First Page Preview
A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 151, Issue 4, 20 February 2011, Pages 295–302
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering