Protein secretion biotechnology using Streptomyces lividans: Large-scale production of functional trimeric tumor necrosis factor alpha

We evaluated the feasibility of large-scale production of biopharmaceutical s expressed as heterologous polypeptides from the Gram-positive bacterium S treptomyces lividans. As a model protein we used murine tumor necrosis fact or alpha (mTNF alpha). mTNF alpha fused C-terminally to the secretory signa l peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature m TNF alpha (80-120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNF alpha from cultu re supernatants was developed. Importantly, the protein is recovered from t he spent growth medium in its native trimeric state as judged by biophysica l analysis. Further, mTNF alpha secreted by S. lividans is significantly mo re active in an in vitro apoptosis tissue culture assay than a correspondin g polypeptide produced in Escherichia coli. This pilot study provides the f irst validation of S. lividans protein secretion as an alternative bioproce ss for large-scale production of oligomeric proteins of potential therapeut ic value. (C) 2001 John Wiley & Sons, Inc.