title: Development of a recombinant bacterial expression system for the active form of a human transforming growth factor beta type II receptor ligand binding domain.
abstract: Expression systems have been designed to test the suitability of expressing the high cysteine containing extracellular domain (residues 1-136) of human transforming growth factor beta type II receptor (TbetaRII). Receptor expressed using a baculovirus system was functional following both enzymatic deglycosylation and elimination of the N-terminal 22 amino acids by protease degradation. Bacterial expression of a TbetaRII lacking the 26 N-terminal amino acids retained the ability to bind its ligand, TGF-beta1. Receptor expressed in bacteria was sensitive to proteolytic degradation at residue Lys98 but a K98T mutation eliminated degradation and did not disrupt binding. Although several different forms of TbetaRII were expressed, only a fusion with glutathione S-transferase gave soluble TbetaRII, which was purified at a yield of 0.1 mg/10 L of bacterial growth. N-Terminal truncations of TbetaRII (residues 22-136 or 27-136) could be refolded from inclusion bodies and purified to an active form with an efficiency of 10%.
authors.affiliation: Structural Biology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, USA.
authors.forename: C C
authors.initials: CC
authors.lastname: Boesen
authors.forename: S A
authors.initials: SA
authors.lastname: Motyka
authors.forename: A
authors.initials: A
authors.lastname: Patamawenu
authors.forename: P D
authors.initials: PD
authors.lastname: Sun
mesh_terms: D001419:Bacteria / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D008024:Ligands; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D017931:DNA Primers; D008024:Ligands; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II