Protein secretion biotechnology usingStreptomyces lividans: Large-scale production of functional trimeric tumor necrosis factor ?

Abstract

We evaluated the feasibility of large‐scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram‐positive bacterium Streptomyces lividans. As a model protein we used murine tumor necrosis factor alpha (mTNFα). mTNFα fused C‐terminally to the secretory signal peptide of the subtilisin‐inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature mTNFα (80–120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNFα from culture supernatants was developed. Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis. Further, mTNFα secreted by S. lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli. This pilot study provides the first validation of S. lividans protein secretion as an alternative bioprocess for large‐scale production of oligomeric proteins of potential therapeutic value. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 611–619, 2001.

Large-scale production of a thermostable Rhodothermus marinus cellulase by heterologous secretion from Streptomyces lividans

“…Streptomyces lividans has been used for the heterologous secretion of several polypeptides of bacterial and eukaryotic origin (for examples see [ 1 – 5 ]). Commonly, heterologous genes are fused to signal peptide sequences from highly expressed/secreted endogenous Streptomyces proteins [ 6 – 8 ].…”

“…Previously, we demonstrated that S. lividans can efficiently secrete active trimeric murine tumor necrosis factor alpha (mTNFα) [ 5 , 10 ], a Jonesia sp. xyloglucanase of 100 kDa [ 1 ] and other polypeptides such as phospholipase D [ 3 ], transglutaminase (TGase), β-1,4-endoglucanase and β-glucosidase [ 11 ] into the growth medium.…”

Metabolomics investigation of recombinant mTNFα production in Streptomyces lividans

“…This is mainly due to S. lividans exhibiting less extracellular proteolytic activity and also a lack of the strong restriction system of S. coelicolor [ 11 – 13 ]. Some important enzymes, as well as recombinant proteins are produced by Streptomyces , including the murine tumour necrosis factor alpha (mTNFα) [ 12 , 14 , 15 ] and human glucagon [ 16 ]. For an overview of recombinant protein production and some recent examples of heterologous (mammalian and microbial) proteins produced using S. lividans as the host organism, as well as how systems biology (including metabolomics) approaches can be developed to improve protein production, the reader is directed to the following excellent reviews [ 17 , 18 ].…”

Secretory production of tetrameric native full-length streptavidin with thermostability using Streptomyces lividans as a host

“…There are a lot of reports concerning the secretory production system using S. lividans as the host [ 13 - 16 ]. The secretory production of useful protein is one of practical ways for industrial protein production due to the simple purification procedures that proteins could be purified directly from the culture broth [ 14 ].…”

“…There are a large number of reports concerning the secretory production of useful proteins using S. lividans as the host. Pozidis et al successfully used it to produce a biopharmaceutical, mTNFa, derived from mouse [ 14 ]. Zhang et al reported the secretory production of human interleukin-4 receptor [ 15 ].…”