Quantification of the physiochemical constraints on the export of spider silk proteins by <it>Salmonella </it>type III secretion

• Main Authors: Voigt Christopher A, Widmaier Daniel M
• Format: Article
• Language: English
• Published: BMC 2010-10-01
• Series: Microbial Cell Factories
• Online Access: http://www.microbialcellfactories.com/content/9/1/78

<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>