:: Volume 17, Issue 2 (Basic Science Issue - February 2015) ::
ZJRMS 2015, 17(2): 47-51 Back to browse issues page
Expression Optimizing and Purification of Recombinant Human Leukemia Inhibitory Factor Produced in E. coli Strain BL21
Houman Kahroba, Hamid Galehdari , Mohammad Shafeei, Saeed Reza Khatami, Ali Khodadadi
Department of Genetics, Shahid Chamran University, Ahvaz, Iran
Abstract:   (13047 Views)
Background : Leukemia inhibitory factor (LIF) is a glycoprotein, categorized as a subfamily of interleukin 6 cytokines which is known in many mammals. A pluripotent cytokine with a wide biological function range has numerous effects on target cells. The LIF regulates neuron survival, hematopoiesis and seen in LIF-/- knockout mice affects blastocyst implantation, also acts as pre-inflammatory cytokine, and regulates immune response. Further, it is able to maintain stem cells poly potency. The main object of present work was expression, optimizing, and purification of recombinant human leukemia inhibitory factor (rhLIF).
Materials and Methods : In this experimental study, Pet28 (+) carrying the LIF gene and kanamycin resistance marker was cloned in E. coli strain BL21. The induction was optimized by altering 3 factors including the temperature, the induction time, and the concentration of the Isopropyl β-D-1-thiogalactopyranoside (IPTG) as inducer. The purification of the recombinant human LIF (rhLIF) was done by single step affinity chromatography. After the purification, method accuracy was proved by Sodium dodecyl sulfate (SDS) -PAGE electrophoresis and Western blotting.
Results : Optimizing of the expression was reached by changing various parameters, and purification has been done successful.
Conclusion : rhLIF undergoes modification by glycosylation to get its full functionality. The produced rhLIF in prokaryotic host in this work is lacking of glycosylation. However, its proper function should be evaluated in further studies.
Keywords: Recombinant human leukemia inhibitory factor, E. coli, Gene expression, Recombinant protein purification
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Type of Study: Research | Subject: Genetics and Molecular Biology

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Volume 17, Issue 2 (Basic Science Issue - February 2015) Back to browse issues page