Three-component secretion system for extracellular delivery of folded proteins in gram-negative bacteria

Background

Efficient production and secretion of functional proteins are essential to a wide range of applications in biotechnology and medicine, from therapeutic protein delivery to industrial enzyme manufacturing. Gram-negative bacteria such as E. coli are commonly used expression hosts due to their rapid growth, affordability, and genetic flexibility.

However, current bacterial secretion systems are limited in their ability to export complex proteins directly into the extracellular environment. Most systems are constrained by cargo size and structural complexity, often failing to secrete folded proteins larger than 10 kilodaltons, especially those with disulfide bonds. These limitations complicate downstream purification and reduce yields, slowing the development of protein-based therapeutics and biomanufacturing platforms.

Technology overview

This technology introduces a three-component secretion system engineered for efficient export of folded proteins from Gram-negative bacteria. The system includes a C39 peptidase-containing ABC transporter (such as CvaB), a membrane fusion protein (such as CvaA), and a TolC-like outer membrane channel. Target proteins are fused to an N-terminal signal motif derived from microcin, enabling secretion of fully folded proteins up to at least 60 kilodaltons, including those with multiple disulfide bonds. The system operates across diverse bacterial hosts, including E. coli and Salmonella, and functions in strains engineered with oxidizing cytoplasms to support disulfide bond formation. It enables the direct secretion of functional nanobodies, cytokines, and enzymes into the culture medium or surrounding environment, offering a streamlined approach for biomanufacturing and in vivo protein delivery.