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Efficient secretion of biologically active mouse tumor necrosis factor alpha by Streptomyces lividans
L Van Mellaert1, C Dillen, P Proost
1Laboratory of Microbiology, Rega Institute, K.U. Leuven, Belgium.
Abstract:
We have studied the production of mouse tumor necrosis factor alpha (mTNF) with Streptomyces lividans as host. mTNF cDNA was fused to the alpha-amylase-encoding gene (aml) of Streptomyces venezuelae ATCC15068 at 12 amino acids (aa) downstream from the signal-peptidase cleavage site so that the aa surrounding this processing site were conserved. S. lividans containing this construct secreted mTNF at moderately high levels (1-10 micrograms/ml) as a biologically active compound of high specific activity (1 x 10(8) units/mg protein). No unprocessed pre-protein and virtually no processed protein could be detected in the cell lysates. N-terminal aa sequence analysis indicated microheterogeneity (-3 to -6 forms) at the N-terminal site of secreted mTNF. It was demonstrated that this microheterogeneity was due to aminopeptidase activity.
Insights
Streptomyces lividans efficiently secreted biologically active mouse tumor necrosis factor alpha (mTNF). This production method utilized a fusion protein strategy, overcoming processing challenges and yielding high-specific-activity mTNF.
Area of Science:
- Biotechnology
- Molecular Biology
- Microbial Engineering
Background:
- Recombinant protein production in microbial hosts is crucial for therapeutic applications.
- Optimizing secretion and processing of foreign proteins in hosts like Streptomyces lividans presents challenges.
- Mouse tumor necrosis factor alpha (mTNF) is a key cytokine with significant biological activity.
Purpose of the Study:
- To engineer Streptomyces lividans for the high-level secretion of biologically active mouse tumor necrosis factor alpha (mTNF).
- To investigate the processing and secretion mechanism of mTNF produced in S. lividans.
- To characterize the specific activity and N-terminal heterogeneity of secreted mTNF.
Main Methods:
- Fusion of mTNF cDNA to the alpha-amylase gene (aml) of Streptomyces venezuelae.
- Expression and secretion of the fusion protein in Streptomyces lividans.
- Quantification of secreted mTNF levels and specific activity assays.
- N-terminal amino acid sequencing of secreted mTNF to analyze processing.
Main Results:
- S. lividans secreted mTNF at moderately high levels (1-10 µg/ml).
- The secreted mTNF exhibited high specific activity (1 x 10^8 units/mg protein) and was biologically active.
- No unprocessed or significantly processed intracellular mTNF was detected.
- N-terminal sequencing revealed microheterogeneity in secreted mTNF, attributed to aminopeptidase activity.
Conclusions:
- Streptomyces lividans is a suitable host for the efficient secretion of biologically active mTNF.
- The fusion strategy effectively directs secretion and ensures biological activity of mTNF.
- Aminopeptidase activity in S. lividans contributes to the observed N-terminal microheterogeneity of secreted mTNF.