title: An efficient method for expression in Escherichia coli and purification of the extracellular ligand binding domain of the human TGFbeta type II receptor.
abstract: 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.
authors.affiliation: Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, 117997 Moscow, Russia. marine gasparian@yahoo.com
authors.forename: Marine E
authors.initials: ME
authors.lastname: Gasparian
authors.forename: Pavel A
authors.initials: PA
authors.lastname: Elistratov
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Yakimov
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: Mikhail P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D001665:Binding Sites; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D053773:Transforming Growth Factor beta1; D013879:Thioredoxins; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II