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

Paper ID Volume ID Publish Year Pages File Format Full-Text
24088 43496 2010 6 PDF Available
Title
An efficient method for expression in Escherichia coli and purification of the extracellular ligand binding domain of the human TGFβ type II receptor
Abstract

TGFβ signaling is initiated by binding of growth factor ligand to two related single-pass transmembrane receptor serine/threonine kinases, known as the TGFβ type I (TβRI) and type II (TβRII-ED) receptors. TβRII-ED is essential for all TGFβ-induced signals. The DNA sequence encoding the extracellular domain of human TβRII-ED (TβRII-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 (∼1 g L−1) of thioredoxin/TβRII-ED fusion was achieved in Escherichia coli BL21(DE3) strain mainly in soluble form. The soluble thioredoxin/TβRII-ED fusion has been purified and refolded on Ni-NTA agarose. After cleavage of purified thioredoxin/TβRII-ED fusion by recombinant human enteropeptidase light chain (L-HEP) the target protein of TβRII-ED was separated from thioredoxin on Ni-NTA agarose. Fourteen milligrams of highly purified TβRII-ED without N- or C-terminal tags was yielded from 100 mL cell culture. The purified preparation of TβRII-ED was highly homogenous, as shown by SDS-PAGE with silver staining, HPLC and mass spectroscopy analysis. The binding of TβRII-ED purified from E. coli to TGFβ1 was shown to be comparable to commercial material purified from NSO cells. Recombinant TβRII-ED could be employed as an antagonist of TGFβ1 and TGFβ3 in vitro and in vivo as well as for therapy of fibrotic disorders and some types of cancer.

Keywords
TGFβ; TGFβ type II receptor; Thioredoxin-tag fusion protein; E. coli; TGFβ targeting
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An efficient method for expression in Escherichia coli and purification of the extracellular ligand binding domain of the human TGFβ type II receptor
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 148, Issues 2–3, 20 July 2010, Pages 113–118
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us