An improved recombinant mammalian cell expression system for human transforming growth factor-β2 and -β3 preparations

Transforming growth factor-β2 and -β3 (TGF-β2 and -β3) are important members of TGF-β 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-β2 and -β3. In this system, we used a mammalian expression vector which contains a glutamine synthetase coding region for amplification, together with a modified TGF-β2 or -β3 open reading frame for expression. The leader peptide of TGF-β2 or -β3 was replaced by that from the V-J2-C region of a mouse immunoglobulin κ-chain, and a poly-histidine tag was inserted immediately after the leader sequence to facilitate protein purification without changing the mature TGF-β2 or -β3 amino acid sequence. In addition, the extreme N-terminal cysteine residue of TGF-β2 or -β3 was replaced by a serine residue. The resulting expression constructs produced two stable cell clones expressing 10 mg of TGF-β2 and 8 mg of TGF-β3 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-β2 and 4 mg of TGF-β3. This method represents a significant improvement over previously published methods and may be applicable to other TGF-β superfamily members. We further confirmed that latent TGF-β2 and -β3 can be activated by proteolysis and glycolysis, which have not been reported before.