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

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Дата последнего поиска статьи во внешних источниках: 18 июля 2013 г.

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[1] An efficient method for expression in escherichia coli and purification of the extracellular ligand binding domain of the human tgfbeta type ii receptor / M. E. Gasparian, P. A. Elistratov, S. A. Yakimov et al. // Journal of Biotechnology. — 2010. — Vol. 148, no. 2-3. — P. 113–118. TGFbeta signaling is initiated by binding of growth factor ligand to two related single-pass transmembrane receptor serine/threonine kinases, known as the TGFbeta type I (TbetaRI) and type II (TbetaRII-ED) receptors. TbetaRII-ED is essential for all TGFbeta-induced signals. The DNA sequence encoding the extracellular domain of human TbetaRII-ED (TbetaRII-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 ( approximately 1 gL(-1)) of thioredoxin/TbetaRII-ED fusion was achieved in Escherichia coli BL21(DE3) strain mainly in soluble form. The soluble thioredoxin/TbetaRII-ED fusion has been purified and refolded on Ni-NTA agarose. After cleavage of purified thioredoxin/TbetaRII-ED fusion by recombinant human enteropeptidase light chain (L-HEP) the target protein of TbetaRII-ED was separated from thioredoxin on Ni-NTA agarose. Fourteen milligrams of highly purified TbetaRII-ED without N- or C-terminal tags was yielded from 100mL cell culture. The purified preparation of TbetaRII-ED was highly homogenous, as shown by SDS-PAGE with silver staining, HPLC and mass spectroscopy analysis. The binding of TbetaRII-ED purified from E. coli to TGFbeta1 was shown to be comparable to commercial material purified from NSO cells. Recombinant TbetaRII-ED could be employed as an antagonist of TGFbeta1 and TGFbeta3 in vitro and in vivo as well as for therapy of fibrotic disorders and some types of cancer.

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