An efficient method for expression in Escherichia coli and purification of the extracellular ligand binding domain of the human TGFbeta type II receptor.
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.
Gasparian
Marine E
ME
Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, 117997 Moscow, Russia. marine gasparian@yahoo.com
Elistratov
Pavel A
PA
Yakimov
Sergey A
SA
Dolgikh
Dmitry A
DA
Kirpichnikov
Mikhail P
MP
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
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