An efficient method for expression in Escherichia coli and purification of the extracellular ligand binding domain of the human TGFβ type II receptor

TGF beta signaling is initiated by binding of growth factor ligand to two related single-pass transmembrane receptor serine/threonine kinases, known as the TGF beta type I (T beta RI) and type II (T beta RII-ED) receptors. T beta RII-ED is essential for all TGF beta-induced signals. The DNA sequence encoding the extracellular domain of human T beta RII-ED (T beta RII-ED, residues 4-136) was synthesized from 20 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. High level expression (similar to 1 g L-1) of thioredoxin/T beta RII-ED fusion was achieved in Escherichia coli BL21(DE3) strain mainly in soluble form. The soluble thioredoxin/T beta RII-ED fusion has been purified and refolded on Ni-NTA agarose. After cleavage of purified thioredoxin/T beta RII-ED fusion by recombinant human enteropeptidase light chain (L-HEP) the target protein of T beta RII-ED was separated from thioredoxin on Ni-NTA agarose. Fourteen milligrams of highly purified T beta RII-ED without N- or C-terminal tags was yielded from 100 mL cell culture. The purified preparation of T beta RII-ED was highly homogenous, as shown by SDS-PAGE with silver staining, HPLC and mass spectroscopy analysis. The binding of T beta RII-ED purified from E. coli to TGF beta 1 was shown to be comparable to commercial material purified from NSO cells. Recombinant T beta RII-ED could be employed as an antagonist of TGF beta 1 and TGF beta 3 in vitro and in vivo as well as for therapy of fibrotic disorders and some types of cancer. (C) 2010 Elsevier B.V. All rights reserved.