Codon optimization, Cloning and expression of the human leukemia inhibitory factor (hLIF) in E. coli

Abstract

Background: Leukemia inhibitor factor (LIF) is a very important pleiotropic cytokine which belongs to interleukin-6 (IL-6) family. LIF exerts multiple effects on different types of cells and tissues with numerous regulatory effects in vivo and in vitro. It is a lymphoid factor, which performs a number of activities including cholinergic neuron differentiation, control of stem cell pluripotency, bone and fat metabolism, and is important for embryo implantation and promoting megakaryocytes production in vivo. Human LIF is a potential therapeutic candidate for some diseases such as multiplesclerosis (MS). Objectives: Because of aforementioned applications of the LIF protein in biological systems, the LIF gene has been cloned in various species. In this study a useful novel method was used to clone an optimized LIF sequence. Materials and Methods: In this study, the optimized cDNA form of human leukemia inhibitory factorwas cloned into the pET-28a (+) expression vector under control of T7lacpromoter using BamHI and XbaI restriction enzymes. The recombinant vector was transformed into Escherichia coli strain BL21 (DE3). The cloned hLIF cDNA was expressed as a fusion protein with His-tag. Cloning of hLIF cDNA was confirmed by digestion and DNA sequencing. Appropriate expression of recombinant hLIF was examined by SDS-PAGE after induction with isopropylthio-β-galactoside (IPTG). Results: The results confirmed the expression of the 19.7 kDa rhLIF protein in the bacterial expression system. Conclusions: We could show that codon optimization might increase the production of recombinant hLIF in the E. coli. This result is useful in similar cases, in which the level of expressed gene is critical. © 2013, National Institute of Genetic Engineering and Biotechnology; Published by Kowsar Corp.