Abstract
Leukemia inhibitory factor [LIF] is a glycoprotein that is able to induce the differentiation of M1 myeloid leukemic cells and prevent the differentiation of murine embryonic stem [ES] cells. This report presents a simple method for the production and purification of large amounts of recombinant murine and human LIF in Escherichia coli. LIF is expressed initially as a fusion product with glutathione Stransferase, separated by a thrombin cleavage site. The fusion product can be rapidly purified on a glutathione-agarose affinity matrix and fully biologically active LIF released from the matrix by cleavage with thrombin. In this form LIF is suitable for many in vitro cell culture uses and in particular for ES cell culture. Further purification to homogeneity of thrombin-released LIF can be achieved by a single fractionation by reversed-phase high performance liquid chromatography; the purified product contains no detectable bacterial endotoxin. Evidence is presented that the intact fusion product has little or no biological activity but biologically active LIF is released by endogenous proteolytic cleavage.
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