The effect of signal sequences on the efficiency of secretion of a heterologous phosphotriesterase by Streptomyces lividans

Rowland, Sharon S.; Zulty, James J.; Sathyamoorthy, Malathi; Pogell, Burton M.; Speedie, Marilyn K.

doi:10.1007/bf00169426

Cited by 16 publications

(7 citation statements)

References 28 publications

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“…Moreover, less manipulation and downstream processing is required to obtain pure protein. The amount of OpdA secreted in our system is similar to that in the S. lividans system (23.6 compared to 21.3 mg/l after 72 h) [Rowland et al, 1992;Steiert et al, 1989], but the B. choshinensis expressed protein is purer, suggesting that very little downstream processing would be needed, mainly to remove media constituents. In the S. lividans system, ammonium sulphate precipitation and then ion exchange chromatography were required to obtain pure protein [Rowland et al, 1992].…”

Section: Discussionsupporting

confidence: 60%

“…The amount of OpdA secreted in our system is similar to that in the S. lividans system (23.6 compared to 21.3 mg/l after 72 h) [Rowland et al, 1992;Steiert et al, 1989], but the B. choshinensis expressed protein is purer, suggesting that very little downstream processing would be needed, mainly to remove media constituents. In the S. lividans system, ammonium sulphate precipitation and then ion exchange chromatography were required to obtain pure protein [Rowland et al, 1992]. This type of purification procedure is all that is required to obtain pure OPH/OpdA from the cytoplasm of E. coli over-expressing these proteins [Yang et al, 2003].…”

Section: Discussionsupporting

confidence: 60%

“…OPH has been successfully secreted in the Gram-positive bacterial species Streptomyces lividans using its own signal peptide, but the majority of the expressed protein was still retained in the cytoplasm of the cell [Steiert et al, 1989]. Changing the signal peptide to the S. lividans ß-galactosidase signal peptide (according to our analysis, a TAT type) greatly enhanced the efficiency of secretion to almost 70% [Rowland et al, 1992]. However, further purification was then required to obtain OPH in a relatively pure form.…”

Section: Introductionmentioning

confidence: 92%

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A <i>Brevibacillus choshinensis</i> System That Secretes Cytoplasmic Proteins

Horne¹,

Williams²,

Sutherland³

et al. 2004

Microb Physiol

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Brevibacillus choshinensis has previously been shown to be a useful strain for the secretion of heterologous proteins via the Sec secretory pathway. This pathway involves the secretion of proteins prior to folding, whereas the alternative TAT (twin-arginine translocation) pathway enables pre-folded proteins to be secreted. We have modified the signal peptide of the Brevibacillus expression vector pNCMO2 to accommodate a Sec avoidance signal as well as the twin arginines required for secretion via the TAT system. Use of this modified signal peptide with the phosphotriesterase OpdA enabled B. choshinensis transformants to express and secrete the enzyme in an active and substantially pure form. The system was also used successfully to secrete two cytoplasmic proteins, the phosphotriesterase HocA from Pseudomonas monteilii and the phenylcarbamate-degrading enzyme, PCD, from Arthrobacter oxydans. The inhibitors carbonyl cyanide m-chlorophenyl hydrazine and sodium azide were used to confirm that secretion was occurring via the TAT secretion pathway. The modified B. choshinensis system we have developed may have general utility in secreting a wide range of heterologous proteins in active and conveniently processed form.

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Section: Discussionsupporting

confidence: 60%

Section: Discussionsupporting

confidence: 60%

Section: Introductionmentioning

confidence: 92%

See 1 more Smart Citation

A <i>Brevibacillus choshinensis</i> System That Secretes Cytoplasmic Proteins

Horne¹,

Williams²,

Sutherland³

et al. 2004

Microb Physiol

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“…strain K30 indicate a lower rate of protein secretion in the recombinant lcp VH2 -and lcp Kb1 -containing strains than in the recombinant lcp K30 -harboring strain. Due to the present signal sequence there are differences in native protein secretion and especially for secretion of heterologous proteins in S. lividans, as previously described (6,14,35,37). These differences in heterologous protein secretion might also explain the different GPC profiles of poly(cis-1,4-isoprene) molecules after incubation with the recombinant lcp VH2 -, lcp Kb1 -, or lcp K30 -harboring strains of S. lividans TK23 (Fig.…”

Section: Discussionmentioning

confidence: 52%

The Genomes of the Non-Clearing-Zone-Forming and Natural-Rubber- Degrading Species Gordonia polyisoprenivorans and Gordonia westfalica Harbor Genes Expressing Lcp Activity in Streptomyces Strains

Bröker

Dietz

Arenskötter

et al. 2008

Appl Environ Microbiol

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The latex-clearing protein (Lcp K30 ) from the rubber-degrading bacterium Streptomyces sp. strain K30 is involved in the cleavage of poly(cis-1,4-isoprene), yielding isoprenoid aldehydes and ketones. Lcp homologues have so far been detected in all investigated clearing-zone-forming rubber-degrading bacteria. Internal degenerated oligonucleotides derived from lcp genes of Streptomyces sp. strain K30 (lcp K30 ), Streptomyces coelicolor strain A3(2), and Nocardia farcinica strains IFM10152 and E1 were applied in PCR to investigate whether lcp homologues occur also in the non-clearing-zone-forming rubber-utilizing bacteria Gordonia polyisoprenivorans strains VH2 and Y2K, Gordonia alkanivorans strain 44187, and Gordonia westfalica strain Kb1, which grow adhesively on rubber. The 1,230-and 1,224-bp lcp-homologous genes from G. polyisoprenivorans strain VH2 (lcp VH2 ) and G. westfalica strain Kb1 (lcp Kb1 ) were obtained after screening genomic libraries by degenerated PCR amplification, and their translational products exhibited 50 and 52% amino acid identity, respectively, to Lcp K30 . Recombinant lcp VH2 and lcp Kb1 harboring cells of the non-rubber-degrading Streptomyces lividans strain TK23 were able to form clearing zones and aldehydes on latex overlay-agar plates, thus indicating that lcp VH2 and lcp Kb1 encode functionally active proteins. Analysis by gel permeation chromatography demonstrated lower polymer concentrations and molecular weights of the remaining polyisoprenoid molecules after incubation with these recombinant S. lividans strains. Reverse transcription-PCR analysis demonstrated that lcp VH2 was transcribed in cells of G. polyisoprenivorans strain VH2 cultivated in the presence of poly(cis-1,4-isoprene) but not in the presence of sodium acetate. Anti-Lcp K30 immunoglobulin Gs, which were raised in this study, were rather specific for Lcp K30 and did not cross-react with Lcp VH2 and Lcp Kb1 . A lcp VH2 disruption mutant was still able to grow with poly(cis-1,4-isoprene) as sole carbon source; therefore, lcp VH2 seems not to be essential for rubber degradation in G. polyisoprenivorans.

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“…The charge balance also supports the presence of PO 4 3-rather than PO 2 F 2 -. There are four six-coordinate (Zn(1), Zn(4), Zn(5) and Zn (8)) and four five-coordinate zinc centres (Zn(2), Zn(3), Zn(6) and Zn(7)) in the complex cation. The molecule is composed of two halves, each composed of two HL1 2-ligands coordinated to four Zn(II) ions.…”

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confidence: 99%