An improved recombinant mammalian cell expression system for human transforming growth factor-beta2 and -beta3 preparations.
Transforming growth factor-beta2 and -beta3 (TGF-beta2 and -beta3) are important members of TGF-beta family which play important roles in the growth, maintenance, and repair processes of developing embryos, neonates, and adults. Preparation of large quantities of these two cytokines, which is necessary for structural studies and other applications, has proven to be extremely difficult. We have developed a novel Chinese hamster ovary cell-based expression system for high-level expression and high recovery of recombinant human TGF-beta2 and -beta3. In this system, we used a mammalian expression vector which contains a glutamine synthetase coding region for amplification, together with a modified TGF-beta2 or -beta3 open reading frame for expression. The leader peptide of TGF-beta2 or -beta3 was replaced by that from the V-J2-C region of a mouse immunoglobulin kappa-chain, and a poly-histidine tag was inserted immediately after the leader sequence to facilitate protein purification without changing the mature TGF-beta2 or -beta3 amino acid sequence. In addition, the extreme N-terminal cysteine residue of TGF-beta2 or -beta3 was replaced by a serine residue. The resulting expression constructs produced two stable cell clones expressing 10 mg of TGF-beta2 and 8 mg of TGF-beta3 per liter of spent medium. The purification scheme involved the use of two simple chromatographic steps with a typical yield of 5 mg of TGF-beta2 and 4 mg of TGF-beta3. This method represents a significant improvement over previously published methods and may be applicable to other TGF-beta superfamily members. We further confirmed that latent TGF-beta2 and -beta3 can be activated by proteolysis and glycolysis, which have not been reported before.
Zou
Zhongcheng
Z
Structural Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, USA.
Sun
Peter D
PD
D000818:Animals; D016466:CHO Cells; D002154:Calpain / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005341:Fibrinolysin / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors; D053781:Transforming Growth Factor beta2 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D053782:Transforming Growth Factor beta3 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D053781:Transforming Growth Factor beta2; D053782:Transforming Growth Factor beta3; D009439:Neuraminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D005341:Fibrinolysin; D002154:Calpain; D002402:Cathepsin D; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase