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Genigraphics® has been producing output from PowerPoint® longer than anyone in the industry; dating back to when we helped Microsoft® design the PowerPoint® software. US and Canada: 1-800-790-4001 Email: info@genigraphics.com [This sidebar area does not print.] The Effect of Protein Folding Kinetics on Substrate Selectivity of the Tat Secretion Pathway in B. subtilis Erin Drufva (PhD student) and Dr. Kang Wu Chemical Engineering Department, University of New Hampshire Erin Drufva University of New Hampshire Chemical Engineering Email: eed2@wildcats.unh.edu Contact 1. Abra Roberts 2. Halie White 3. Ryan McEachern 4. Sachi Nagada Acknowledgements Bacillus subtilis is a Gram-positive soil bacterium that secretes numerous enzymes, the production of which represents about 60% of the industrial-enzyme market, as secreting proteins into the extracellular media can greatly reduce downstream processing costs. However, about one third of industrial proteins need to be expressed heterologously and misfolding is often a problem in secretory production of these proteins. The Sec (Secretory) and Tat (Twin arginine translocation) pathways are two major secretion pathways identified in B. subtilis. The Sec pathway secretes the majority of proteins as peptides in an unfolded form while the Tat pathway targets and translocates folded proteins. Signal Peptides (SP) Sec SP Tat SP Both Sec and Tat substrates possess a N-terminal signal peptide (SP) with a similar three-part structure, but the Tat SPs are often longer and have a conserved pattern with two arginine residues. Many proteins with a Tat SP are secreted through the Sec pathway and some Sec substrates, under certain conditions, are secreted by the Tat pathway. The crosstalk between the two pathways suggests that SPs, though very important to direct the proteins to the translocases, are not the only determinant. The aim of this project is to identify other factors that contribute to the substrate selectivity of the Tat pathway to aid in the rational design of heterologous proteins for functional secretion through this pathway. Introduction Methods Experimental Design Four GFPs (Green Fluorescent Proteins) with similar structure and surface properties will be expressed from the same regulatory elements to compare their folding rates in B. subtilis. 60 strains will be constructed to compare the secretion efficiency of proteins with different folding rates via the Tat pathway: Four GFPs (Green Fluorescent Proteins) with similar structure and surface properties but different folding kinetics: eGFP, sfGFP, GFPmut3, and GFPmut3* Three Tat SPs: TorAsp, PhoDsp, and YwbNsp Five host backgrounds: Wildtype, ΔSec, ΔTat, ΔTatAdCd, and ΔTatAyCy Characterization Kinetic measurements of fluorescence from B. subtilis cells expressing each of the four GFPs with a fluorescence microplate reader to compare their folding rates. Fluorescence measurements of the 60 strains and GFP localization analysis using a combination of fluorescence microplate reader, fluorescence microscopy, cell fractionation, and western blotting. Our results confirm that these four GFPs have different folding rates. They can be used to test our hypothesis. GFPs fused with PhoDsp have been constructed and integrated into the wildtype B. subtilis. TorAsp and YwbNsp are being fused to the GFPs. After all strains are constructed, the effect of folding rate on Tat substrate selectivity will be tested. Conclusions and Future Work Results S/T-R-R-X-Φ-Φ Hypothesis Proteins that fold faster are more likely to be targeted and translocated by the Tat pathway. Sec vs. Tat Tat signal peptide Sec signal peptide Sec translocaseTat translocase pDR111 gfp TorAsp-gfp pDR111 Phyper-spank