Quantification of the physiochemical constraints on the export of spider silk proteins by <it>Salmonella </it>type III secretion
<p>Abstract</p> <p>Background</p> <p>The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in <it&...
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doaj-04b2cba2cd4b4884adf0419281dd86bb2020-11-25T00:37:48ZengBMCMicrobial Cell Factories1475-28592010-10-01917810.1186/1475-2859-9-78Quantification of the physiochemical constraints on the export of spider silk proteins by <it>Salmonella </it>type III secretionVoigt Christopher AWidmaier Daniel M<p>Abstract</p> <p>Background</p> <p>The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in <it>Salmonella </it>Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein.</p> <p>Results</p> <p>To test how the timing and level of protein expression affects secretion, we designed a hybrid promoter that combines an IPTG-inducible system with a natural genetic circuit that controls effector expression in <it>Salmonella </it>(<it>psicA</it>). LacO operators are placed in various locations in the <it>psicA </it>promoter and the optimal induction occurs when a single operator is placed at the +5nt (234-fold) and a lower basal level of expression is achieved when a second operator is placed at -63nt to take advantage of DNA looping. Using this tool, we find that the secretion efficiency (protein secreted divided by total expressed) is constant as a function of total expressed. We also demonstrate that the secretion flux peaks at 8 hours. We then use whole gene DNA synthesis to construct codon optimized spider silk genes for full-length (3129 amino acids) <it>Latrodectus hesperus </it>dragline silk, <it>Bombyx mori </it>cocoon silk, and <it>Nephila clavipes </it>flagelliform silk and PCR is used to create eight truncations of these genes. These proteins are all unfolded polypeptides and they encompass a variety of length, charge, and amino acid compositions. We find those proteins fewer than 550 amino acids reliably secrete and the probability declines significantly after ~700 amino acids. There also is a charge optimum at -2.4, and secretion efficiency declines for very positively or negatively charged proteins. There is no significant correlation with hydrophobicity.</p> <p>Conclusions</p> <p>We show that the natural system encoded in SPI-1 only produces high titers of secreted protein for 4-8 hours when the natural <it>psicA </it>promoter is used to drive expression. Secretion efficiency can be high, but declines for charged or large sequences. A quantitative characterization of these constraints will facilitate the effective use and engineering of this system.</p> http://www.microbialcellfactories.com/content/9/1/78 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Voigt Christopher A Widmaier Daniel M |
spellingShingle |
Voigt Christopher A Widmaier Daniel M Quantification of the physiochemical constraints on the export of spider silk proteins by <it>Salmonella </it>type III secretion Microbial Cell Factories |
author_facet |
Voigt Christopher A Widmaier Daniel M |
author_sort |
Voigt Christopher A |
title |
Quantification of the physiochemical constraints on the export of spider silk proteins by <it>Salmonella </it>type III secretion |
title_short |
Quantification of the physiochemical constraints on the export of spider silk proteins by <it>Salmonella </it>type III secretion |
title_full |
Quantification of the physiochemical constraints on the export of spider silk proteins by <it>Salmonella </it>type III secretion |
title_fullStr |
Quantification of the physiochemical constraints on the export of spider silk proteins by <it>Salmonella </it>type III secretion |
title_full_unstemmed |
Quantification of the physiochemical constraints on the export of spider silk proteins by <it>Salmonella </it>type III secretion |
title_sort |
quantification of the physiochemical constraints on the export of spider silk proteins by <it>salmonella </it>type iii secretion |
publisher |
BMC |
series |
Microbial Cell Factories |
issn |
1475-2859 |
publishDate |
2010-10-01 |
description |
<p>Abstract</p> <p>Background</p> <p>The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in <it>Salmonella </it>Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein.</p> <p>Results</p> <p>To test how the timing and level of protein expression affects secretion, we designed a hybrid promoter that combines an IPTG-inducible system with a natural genetic circuit that controls effector expression in <it>Salmonella </it>(<it>psicA</it>). LacO operators are placed in various locations in the <it>psicA </it>promoter and the optimal induction occurs when a single operator is placed at the +5nt (234-fold) and a lower basal level of expression is achieved when a second operator is placed at -63nt to take advantage of DNA looping. Using this tool, we find that the secretion efficiency (protein secreted divided by total expressed) is constant as a function of total expressed. We also demonstrate that the secretion flux peaks at 8 hours. We then use whole gene DNA synthesis to construct codon optimized spider silk genes for full-length (3129 amino acids) <it>Latrodectus hesperus </it>dragline silk, <it>Bombyx mori </it>cocoon silk, and <it>Nephila clavipes </it>flagelliform silk and PCR is used to create eight truncations of these genes. These proteins are all unfolded polypeptides and they encompass a variety of length, charge, and amino acid compositions. We find those proteins fewer than 550 amino acids reliably secrete and the probability declines significantly after ~700 amino acids. There also is a charge optimum at -2.4, and secretion efficiency declines for very positively or negatively charged proteins. There is no significant correlation with hydrophobicity.</p> <p>Conclusions</p> <p>We show that the natural system encoded in SPI-1 only produces high titers of secreted protein for 4-8 hours when the natural <it>psicA </it>promoter is used to drive expression. Secretion efficiency can be high, but declines for charged or large sequences. A quantitative characterization of these constraints will facilitate the effective use and engineering of this system.</p> |
url |
http://www.microbialcellfactories.com/content/9/1/78 |
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