Expression of recombinant human soluble type II transforming growth factor-beta receptor in Pichia pastoris and Escherichia coli: two powerful systems to express a potent inhibitor of transforming growth factor-beta.
Transforming growth factor-beta (TGF-beta) is a potent regulator of cell metabolism, proliferation, and differentiation. To study the role of endogenous TGF-beta in processes such as tissue repair and inflammation, potent and specific inhibitors are required. Because the type II TGF-beta receptor (TGF beta RII) has a high affinity for TGF-beta, the extracellular domain of TGF beta RII (TGF-beta sRII) was expressed in Pichia pastoris and Escherichia coli. Expression of the soluble TGF beta sRII using P. pastoris resulted in a soluble, heterogeneously glycosylated protein which was secreted into the medium. Although expression of TGF beta sRII in E. coli resulted in the formation of insoluble inclusion bodies, solubilization and refolding resulted in a biologically active protein. Because in both systems a C-terminal 6x His coding sequence was inserted behind the coding sequence for the extracellular domain of TGF beta RII the recombinant proteins could be purified by a powerful, single-step procedure using a Ni-NTA agarose. The purified proteins appeared to be potent inhibitors of TGF-beta 1 and TGF-beta 3. In contrast, TGF beta sRII was less effective in neutralization of TGF-beta 2. In conclusion, biologically active TGF beta sRII can be produced using P. pastoris and E. coli expression systems. The ease of these expression systems, the powerful single step purification and low costs makes it possible to produce TGF beta s RII in large amounts to further elucidate the role of TGF-beta 1 and TGF-beta 3 in physiological processes like tissue repair and inflammation.
Glansbeek
H L
HL
Department of Rheumatology, University Hospital Nijmegen, The Netherlands.
van Beuningen
H M
HM
Vitters
E L
EL
van der Kraan
P M
PM
van den Berg
W B
WB
D001483:Base Sequence; D001681:Biological Assay; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D006801:Humans; D009994:Osmolar Concentration; D010843:Pichia / Q000235:genetics; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013936:Thymidine / Q000032:analysis / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014316:Tritium
D017931:DNA Primers; D018076:DNA, Complementary; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D014316:Tritium; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D013936:Thymidine