(6go) Recovery of Folded Heterologous Proteins in the Extracellular Space from Bacterial Culture

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

Heterologous protein production in bacteria is a batch process, where the cells are lysed and the protein of interest is purified from the cellular milieu. Protein secretion into the extracellular space simplifies protein purification and enables continuous processing. We use synthetic biology and protein engineering techniques to optimize a bacterial strain for production and secretion of heterologous proteins of interest (POIs). We start by engineering the apparatus of the type III secretion system (T3SS) of Salmonella enterica, a heterogeneous multi-protein structure that spans the periplasm, inner and outer membranes, and projects out from the cell, resembling a hypodermic needle.

To increase secreted protein titers, we must control the expression of the secretion apparatus. Critically, expression of the genes that code for the apparatus is tightly regulated and very sensitive, and native expression of these genes is low and only occurs in a fraction of the culture. We achieve a greater than 10-fold increase in secreted protein titer, exceeding 10 mg/L, using transcriptional control. We control the expression of the positive transcription factor, HilA, the native regulator of the apparatus genes. By synthetically controlling expression of the apparatus genes, we increase expression of the apparatus genes and activate nearly all the cells in the culture for secretion. This approach is generalized for all proteins tested, as it does not manipulate the POI.

Additionally, we characterize the folding of the secreted protein product. 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. Spontaneous refolding in the extracellular space is desired for recovery of correctly folded proteins. 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 diverse models proteins. Standard functional assays indicate that these proteins refold into functional forms after secretion. In addition, our data suggests that post-translational modifications, such as metal cofactor biding, multimerization, and disulfide bond formation, spontaneously occur post-secretion. Together, recovery of folded heterologous protein at high titer in the extracellular space exhibits a strength of the use of a protein secretion chassis.

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