Overexpression of human transforming growth factor-beta1 using a recombinant CHO cell expression system
| Autor: | Peter D. Sun, Zhongcheng Zou |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Signal peptide
Blotting Western Cell Culture Techniques Enzyme-Linked Immunosorbent Assay Plasma protein binding CHO Cells Biology Protein Sorting Signals Biochemistry Models Biological Mass Spectrometry law.invention Cell Line Transforming Growth Factor beta1 law Transforming Growth Factor beta Cricetinae Protein purification Animals Humans Histidine Cysteine Disulfides Serum Albumin Expression vector Dose-Response Relationship Drug Cell growth Chinese hamster ovary cell Molecular biology Recombinant Proteins Rats Cell culture Recombinant DNA Electrophoresis Polyacrylamide Gel Biotechnology Plasmids Protein Binding |
| Zdroj: | Protein expression and purification. 37(2) |
| ISSN: | 1046-5928 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect