(570a) Refolding of Heterologous Proteins in the Extracellular Space after Secretion

Authors: 
Metcalf, K. J., University of California, Berkeley
Rosales, S., University of California, Berkeley
Azam, A., University of California Berkeley
Valdivia, E., University of California, Berkeley
Tullman-Ercek, D., University of California, Berkeley

Recombinant DNA technology enables the production of pure, folded heterologous proteins, where a gene coding for a protein of interest (POI) is expressed in a host cell. However, this process requires significant optimization for each new POI; heterologous protein production in bacteria is often a batch process, in which the cells are lysed and the POI is purified from the cellular milieu, sometimes requiring refolding. One strategy to simplify protein purification and enable continuous processing is to secrete the POI directly into the extracellular space. Toward this goal, we use synthetic biology and protein engineering techniques to optimize a bacterial strain for production and secretion of heterologous proteins. We engineer the type III secretion system (T3SS) of Salmonella enterica, a heterogeneous multi-protein structure that secretes proteins from the cytoplasm to the extracellular space in one step. In this work, we hypothesized that the secreted protein product would refold in the extracellular space. Recent structural data shows that the protein is unfolded and linearized as it is secreted through the “needle”, which is 2 nm in diameter and 50 nm long. We use enzymatic activity as a proxy for protein folding, and demonstrate enzyme activity in the extracellular space, indicating that secreted proteins indeed refold after secretion. Genetic and chemical methods are used to probe the folded state of the model enzymes beta-lactamase and alkaline phosphatase and a single-chain variable fragment (scFv) from an antibody. Standard functional assays indicate that the enzymes and scFvs refold into functional forms after secretion and spontaneous form the requisite post-translational modifications (eg. disulfide bond formation, metal ion cofactor binding, multimerization). Recovery of folded heterologous protein in the extracellular space exhibits a strength of the use of a protein secretion chassis.