275821 Quantitative Functional Single-Cell Characterization of Surface Display of Recombinant Proteins Using A Bacterial Autotransporter

Monday, October 29, 2012: 2:36 PM
Fayette (Westin )
Balakrishnan Ramesh, Chemical and Biomolecular Engineering, University of Houston, Houston, TX, Patrick C. Cirino, Chemical & Biomolecular Engineering, University of Houston, Houston, TX and Navin Varadarajan, Dept. of Chemical & Biomolecular Engineering, University of Houston, Houston, TX

Quantitative functional single-cell characterization of surface display of recombinant proteins using a bacterial autotransporter

Balakrishnan Ramesh, Patrick C. Cirino, Navin Varadarajan

University of Houston, S222 Engineering Bldg 1, Houston, TX, 77204-4004, USA

Expressing functional recombinant proteins on the surface of bacteria has generated substantial interest due to its utility in biotechnological applications such as whole-cell biocatalysis, bioremediation, responsive biosensors and protein engineering. The mostly widespread protein family in nature that facilitates surface display of native proteins is the autotransporter (AT) pathway. ATs are ubiquitous in Gram negative bacteria and facilitate the translocation and secretion/ display of diverse passenger proteins with functions such as adhesion, proteolysis, and pore formation. ATs comprise of an extended N-terminal leader sequence cleaved at the inner membrane followed by an N-terminal passenger (20-400 kDa) typically associated with virulence functions and a conserved C-terminal ~30kDa β-barrel. Replacing native passenger domain with heterologous protein facilitates the surface display of large proteins at a high number of copies per cell. However, there exists considerable controversy in the ability of ATs to transport passengers, either native or recombinant, containing folded elements, especially those containing disulfide bonds.

We have systematically investigated the ability of the E. coli AT Antigen 43 (Ag43) to display two different recombinant reporter proteins, a single-chain antibody (scFv) that contains two disulfides and chymotrypsin that contains four disulfides. Using flow-cytometry to quantify surface display of functional protein at the single-cell level, we demonstrate that in spite of the known propensity of these passengers to fold in the periplasm, surface display of these proteins is rather efficient and can be achieved using  only the C-terminal domain containing the α-helix and the β-barrel. Our flow-cytometric data is consistent with data obtained via either fluorescence microscopy or western blotting on fractionated cell-compartments. In contrast to previous reports, no genetic manipulation like the use of ompT- strains or the use of reducing agents like β-mercaptoethanol was necessary to accomplish efficient display. Our results indicate that display of recombinant proteins containing multiple disulfides can be achieved by employing the Ag43 system and that the vast majority of native AT including the autochaperone domain are not indispensable for heterologous protein display. We anticipate that this methodology will be especially useful for the recombinant display and screening of protease libraries for a broad variety of applications.

Extended Abstract: File Not Uploaded