High-level Soluble Expression, Purification, and Functional Characterization of the Recombinant Human Leukemia Inhibitory Factor: A Potential General Strategy for the Recombinant Expression of Cytokines Consisting of Four α-Helices in a Bundle

Jie      Lin; Jianjun      Liu; Minnan      Lu; Shuangsheng      Deng; Lan      Ma

doi:10.2174/092986611795446058

Abstract

The human leukemia inhibitory factor (hLIF) is one of the most important cytokines in the interleukin-6 (IL-6) cytokine family. Numerous studies have demonstrated that hLIF is a pleiotropic cytokine with multiple effects on different types of cells and tissues. The optimal chemical synthesis of the hLIF gene has been previously reported to increase the expression of the recombinant inclusion body protein in E. coli. However, the required refolding step limits the recovery rate. In this report, a novel strategy was designed to produce a soluble recombinant human LIF (rhLIF) in the prokaryotic system in order to obtain higher yields of the bioactive protein with simpler steps. This optimal hLIF gene was cloned, and it successfully expressed the soluble recombinant protein in E. coli using the thioredoxin (Trx) protein as a fusion partner. A simple purification procedure is established to purify the recombinant fusion protein from the soluble supernatant of the lysed culture cells. This procedure yields up to 5 mg/L rhLIF with above 95% purity. The strategy allows the protease to release target cytokines without additional N-terminus amino acids, which is an important consideration for maintaining its bioactivity. Functional analysis of the purified rhLIF by murine myeloblastic leukemia M1 cell proliferation assay demonstrates biological activity that is similar and comparable to that of hLIF. These results present a sound strategy for the soluble production of rhLIF and other homologous tertiary structure cytokines consisting of four ..-helices in a bundle for basic research, as well as clinical applications.

Keywords: Enterokinase release, human leukemia inhibitory factors, functional characterization, fusion partners, thioredoxin protein, simple purification, soluble expressionEnterokinase release, human leukemia inhibitory factors, functional characterization, fusion partners, thioredoxin protein, simple purification, soluble expression