Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner

Abstract

CitationPreparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner. This is a pre-or post-print of an article published in Tomala, M., Lavrentieva, A., Moretti, P., Rinas, U., Kasper, C., Stahl, F., Schambach, A., Warlich, E., Martin, U., Cantz, T., Scheper, T. Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major costs (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant E. coli cultures. hLIF was cloned into pET32b-trx and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein underwent cleavage by specific TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of low soluble proteins through expression in fusion with the solubilizing partner thioredoxin.

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

“…The hLIF fusion proteins purities were greater than 95% (Table 1). The yields of His 6 -hLIF and Trx-His 6 -hLIF fusion proteins in this work were higher than previously reported (Gearing et al, 1989;Samal et al, 1995;Tomala et al, 2010).…”

“…Many studies have successfully used Escherichia coli to produce several human growth factors and cytokines, such as interleukin-1 (Furutani et al, 1985), interleukin-4 (van Kimmenade et al, 1988), epidermal growth factor (Shimizu et al, 1991;Abdull Razis et al, 2008), fibroblast growth factor (Squires et al, 1988;Gasparian et al, 2009) and LIF (Gearing et al, 1989;Samal et al, 1995;Tomala et al, 2010) for therapeutic and clinical purposes. Although, recombinant human LIF (hLIF) is available and has been successfully produced in E. coli expression systems, they still require many steps of downstream processing.…”

“…Although, recombinant human LIF (hLIF) is available and has been successfully produced in E. coli expression systems, they still require many steps of downstream processing. In addition, these systems have also shown multiple disadvantages: (1) high amounts of affinity resins are needed, (2) expensive proteases are required to cleave the target protein from the affinity tag, and (3) additional chromatography steps are needed to remove the proteases (Gearing et al, 1989;Tomala et al, 2010). From these reasons, these methods are not cost effective for long-term production of recombinant proteins.…”

Simple and efficient expression of codon-optimized mouse leukemia inhibitory factor in <i>Escherichia coli</i>

“…The cells play a primary role in cell therapies and tissue replacement therapies [6,7]. Recently, rhLIF was successfully produced and purified by several groups using different kinds of expression systems such as E. coli [8][9][10], yeast cells and even in rice [11]. An issue arising from this process is the soaring cost of LIF and other cytokines in pluripotent stem cells research.…”

“…An issue arising from this process is the soaring cost of LIF and other cytokines in pluripotent stem cells research. Numerous studies have suggested that the cost of commercially produced LIF accounts for about 90 % pluripotent stem cells culture medium [8]. It is urgent to obtain various forms of LIF in high quantities and at a low cost and biological active.…”