Overexpression of human transforming growth factor-beta1 using a recombinant CHO cell expression system.
Transforming growth factor-beta1 (TGF-beta1) is secreted by most cells as a high molecular weight latent complex, which consists of latent TGF-beta1 disulfide bonded to latent TGF-beta1-binding protein (LTBP). Current recombinant expression systems yield less than 1-2 mg of the mature TGF-beta1 per liter of cell culture medium. In an effort to produce large quantities of the recombinant cytokine for structural studies, we have constructed a mammalian expression system based on a modified pcDNA3.1(+) vector with a glutamine synthetase gene inserted for gene amplification. The leader peptide of TGF-beta1 was replaced with that of rat serum albumin, and an eight-histidine tag was inserted immediately after the leader sequence to facilitate protein purification. In addition, Cys 33 of TGF-beta1, which forms a disulfide bond with LTBP, was replaced by a serine residue. The resulting expression construct produced a stable clone expressing 30 mg of mature TGF-beta1 per liter of spent medium. Purified TGF-beta1 bound with high affinity to its type II receptor with a solution dissociation constant of approximately 70 nM, and was fully active in both a Mv1Lu cell growth inhibition assay and in a PAI-1 luciferase reporter assay. Owing to similarities in the synthesis, secretion, and structure of TGF-beta family members, this recombinant expression system may also be applied to the overexpression of other TGF-beta isomers and even to members of the TGF-beta superfamily to facilitate their preparation.
Zou
Zhongcheng
Z
Structural Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
Sun
Peter D
PD
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D016466:CHO Cells; D018929:Cell Culture Techniques; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006639:Histidine / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry; D016212:Transforming Growth Factor beta / Q000096:biosynthesis*; D053773:Transforming Growth Factor beta1
D004220:Disulfides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012709:Serum Albumin; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D006639:Histidine; D003545:Cysteine