425236 Directed Co-Evolution of the Twin-Arginine Translocation Machinery for Enhanced Export of Recombinant Proteins

Wednesday, November 11, 2015: 3:33 PM
151A/B (Salt Palace Convention Center)
May Taw, Microbiology Graduate Program, Cornell University, Ithaca, NY, Mark A. Rocco, Biomedical Engineering, Cornell University, Ithaca, NY and Matthew P. DeLisa, Chemical & Biomolecular Engineering, Cornell University, Ithaca, NY

­­Directed co-evolution of the twin-arginine translocation machinery for enhanced export of recombinant proteins


May N. Taw1,2, Mark A. Rocco2, and Matthew P. DeLisa2

1. Microbiology Graduate Program, Cornell University, Ithaca, NY

2. Department of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY


Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. Often it is desired to secrete recombinant proteins into the periplasmic space as a means to simplify downstream purification and increase protein stability by avoiding proteolytic degradation in the cytoplasm. The predominant protein translocation mechanism in E. coli is the general secretory (Sec) pathway; however, a wealth of information has emerged over the past decade on an alternative, Sec-independent protein export mechanism known as the twin-arginine translocation (Tat) pathway. The hallmark of the Tat system is its unique ability to export already folded proteins to the periplasmic space. Our group and others have shown that the Tat machinery has an in-built quality-control mechanism that discriminates the folding state of substrate proteins, allowing only folded proteins to transit while rejecting misfolded substrates from the translocation cycle. However, despite its potential for localizing correctly folded proteins in the periplasm, translocation by the Tat system is approximately an order of magnitude slower compared to the Sec pathway. To address this issue, we engineered the Tat translocase, comprised of the TatABC integral membrane proteins, to hyper-secrete industrially relevant proteins into the periplasm using a directed co-evolution approach. This involved generation of a combinatorial tatABC library, followed by genetic selection of translocase variants capable of increased export of a single-chain Fv (scFv) antibody fragment specific for β-galactosidase called scFv13-R4. Following just a single round of mutagenesis and selection, we isolated five unique TatABC hyper-secretors that exhibited export efficiency that was greatly enhanced compared to the parental Tat machinery and also compared to Sec pathway. The selected mutants carried substitutions in TatB and TatC, but not TatA, implicating these proteins as key regulators of protein flux to the periplasm. We conclude that substrate “proofreading” is a rate-limiting step in the translocation cycle given that the quality-control mechanism of all hyper-secretors was found to be relaxed. Taken together, our results provide new insights on a poorly understood aspect of the Tat transport mechanism and at the same time furnish a set of optimized Tat translocases capable of high levels of recombinant protein export.

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