Efficient secretion of biologically active mouse tumor necrosis factor α by Streptomyces lividans

doi:10.1016/0378-1119(94)90876-1

Gene

Volume 150, Issue 1, 2 December 1994, Pages 153-158

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Abstract

We have studied the production of mouse tumor necrosis factor α (mTNF) with Streptomyces lividans as host. mTNF cDNA was fused to the α-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 μg/ml) as a biologically active compound of high specific activity (1 × 10⁸ 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.

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Cited by (30)

Functional analysis of TatA and TatB in Streptomyces lividans
2005, Biochemical and Biophysical Research Communications
Citation Excerpt :
S. lividans TK24 and its derivatives were precultured in 5 ml phage medium [14] supplemented with thiostrepton (50 μg/ml), apramycin (50 μg/ml) or kanamycin (50 μg/ml), if necessary, and grown at 27 °C with continuous shaking at 300 rpm for 48 h. After homogenization of the mycelium, the strains were inoculated in liquid NM medium [15]. For solid medium, MRYE was used [16] supplemented with thiostrepton (50 μg/ml), apramycin (50 μg/ml) or kanamycin (50 μg/ml), if applicable.
Recently, genes encoding TatA, TatB, and TatC homologues were identified in Streptomyces lividans and the functionality of the twin-arginine translocation (Tat) pathway was demonstrated. Previously, we have shown that TatC is indispensable for Tat-dependent secretion in S. lividans. In the present work, we demonstrate that as TatB, S. lividans TatA is important but not essential for efficient secretion of xylanase C and tyrosinase. The results presented here indicate that in the presence of TatC, still partially functional translocation systems composed of TatAC or TatBC can be formed, suggesting that TatA and TatB have at least partially overlapping activities. However, the dissimilar effect caused by a tatA deletion or a tatB deletion on Tat-dependent secretion together with the fact that TatA cannot fully functionally substitute TatB and vice versa indicates that in S. lividans TatA and TatB are not functionally equivalent. Interestingly, soluble GST-tagged TatA and TatB were able to specifically bind Tat-dependent preproteins. The ability to bind Tat-dependent preproteins together with their cytoplasmic localization in S. lividans strongly suggests that both TatA and TatB, independently or associated, serve to recruit Tat-dependent preproteins to the translocase.
Structural organization of the twin-arginine translocation system in Streptomyces lividans
2005, FEBS Letters
The twin-arginine translocation (Tat) system exports folded proteins across bacterial cytoplasmic membranes. Recently, genes encoding TatA, TatB and TatC homologues were identified in Streptomyces lividans and the functionality of the Tat pathway was demonstrated. Here, we have examined the localization and structural organization of the Tat components in S. lividans. Interestingly, besides being membrane-associated proteins, S. lividans TatA and TatB were also detected in the cytoplasm. TatC could only be detected in isolated membrane fractions. Whereas all TatC was found to be stably inserted in the membrane, part of membrane-associated TatA and TatB could be extracted following high salt, sodium carbonate or urea treatment suggesting a more loose association with the membrane. Finally, we have analyzed Tat complexes that could be purified from an S. lividans TatABC overproducing strain. From the cytoplasmic membrane, two types of high molecular mass Tat complexes could be isolated having a similar composition as those isolated from Escherichia coli. In the cytoplasm, TatA and TatB were detected as monomer or as homo-oligomeric complexes.
Comparison of the Sec and Tat secretion pathways for heterologous protein production by Streptomyces lividans
2004, Journal of Biotechnology
Streptomyces is an interesting host for the secretory production of recombinant proteins because of its natural ability to secrete high levels of active proteins into the culture broth and the availability of extensive fermentation knowledge. In bacterial expression systems, heterologous protein secretion has, so far, almost exclusively been investigated using signal peptides that direct the secretion to the Sec pathway. In this study, we assessed the possibility of the Streptomyces lividans twin-arginine translocation (Tat) pathway to secrete the human proteins tumor necrosis factor (TNF) α and interleukin (IL) 10 by fusing the coding sequences of mature hTNFα and hIL10 to the twin-arginine signal peptides of S. lividans xylanase C (XlnC) and Streptomyces antibioticus tyrosinase. Both proteins were secreted and this secretion was blocked in the ΔtatB and ΔtatC single mutants, indicating that the transport of hTNFα and hIL10 could be directed through the Tat pathway. Secretion levels of hTNFα and hIL10, however, were lower for Tat-dependent than for Sec-dependent transport using the Sec-dependent signal peptide of the Streptomyces venezuelae subtilisin inhibitor. Surprisingly, Sec-dependent transport was enhanced in the tatB deletion strain. This was especially interesting in the case of hIL10, where Sec-dependent transport of hIL10 was at least 15 times higher in the ΔtatB mutant than in the wild-type strain.
Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66
1997, Protein Expression and Purification
A gene encoding the extracellular domain of the human erythropoietin receptor (EPO-R) was constructed using oligonucleotides, with a view to maintaining preferred codon usage for the Streptomycetes. The gene was subcloned into a multicopyStreptomyces–Escherichia colishuttle vector, pCAN46 (derived from pIJ680), containing a strong constitutive promoter from theS. fradiae aphgene, a signal peptide coding region derived from the protease B gene ofS. griseus,and a transcription terminator sequence also derived from theS. fradiae aphgene. Extracellular expression of authentic EPO-R byS. lividanswas demonstrated using SDS–PAGE and Western blot analysis, followed by direct amino terminal sequencing of the purified product. Specific binding ofS. lividans-expressed EPO-R to recombinant human glycosylated EPO was demonstrated using BIAcore (surface plasmon resonance) analysis and native gel shift assays.
Expression by Streptomyces lividans of the rat α integrin CD11b A-domain as a secreted and soluble recombinant protein
2007, Journal of Biomedicine and Biotechnology
Metabolomics investigation of recombinant mTNFaα production in Streptomyces lividans
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Abstract

References (19)

Streptomyces lividans as host for heterologous protein production

FEMS Microbiol. Lett.

Secretory synthesis of human interleukin-2 by Streptomyces lividans

Gene

Streptomyces: a host for heterologous gene expression

Curr. Opin. Biotechnol.

Cloning and characterization of an aminopeptidase P-encoding gene from Streptomyces lividans

Gene

Co valent linking of Fmoc amino acids to resin for solid phase peptide synthesis

Tetrahedron Lett.

Analysis of the structure-function relationship of tumour necrosis factor. Human/mouse chimeric TNF proteins: general properties and epitope analysis

J. Mol. Biol.

Cloning, characterization and regulation of an α-amylase gene from Streptomyces venezuelae

Gene

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Gene

Purification and properties of an extracellular aminopeptidase from Streptomyces lividans 1326

J. Gen. Microbiol.

Cited by (30)

Functional analysis of TatA and TatB in Streptomyces lividans

Structural organization of the twin-arginine translocation system in Streptomyces lividans

Comparison of the Sec and Tat secretion pathways for heterologous protein production by Streptomyces lividans

Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66

Expression by Streptomyces lividans of the rat α integrin CD11b A-domain as a secreted and soluble recombinant protein

Metabolomics investigation of recombinant mTNFaα production in Streptomyces lividans