Abstract
The demand for recombinant proteins for analytic and therapeutic purposes is increasing; however, most currently used bacterial production systems accumulate the recombinant proteins in the intracellular space, which requires denaturating procedures for harvesting and functional testing. We here present a novel FimH-based expression system that enables display of fully functional eukaryotic proteins while preventing technical difficulties in translocating, folding, stabilizing and isolating the displayed proteins. As examples, Gaussia Luciferase (GLuc), epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and epiregulin (EPRG) were expressed as FimH fusion proteins on the surface of E. coli bacteria. The fusion proteins were functionally active and could be released from the bacterial surface by specific proteolytic cleavage into the culture supernatant allowing harvesting of the produced proteins. EGFR ligands, produced as FimH fusion proteins and released by proteolytic cleavage, bound to the EGF receptor (EGFR) on cancer cells inducing EGFR phosphorylation. In another application of the technology, GLuc-FimH expressed on the surface of bacteria was used to track tumor-infiltrating bacteria by bioluminescence imaging upon application to mice, thereby visualizing the colonization of transplanted tumors. The examples indicate that the FimH-fusion protein technology can be used in various applications that require functionally active proteins to be displayed on bacterial surfaces or released into the culture supernatant.
Highlights
Bacterial surface display of recombinant proteins has become an attractive strategy for a broad range of applications such as production of bioadsorbents[1], generation of cellular biosensors[2], development of novel vaccine platforms[3], screening of antibody libraries[4] and whole-cell biocatalysis[5]
Upon IPTG treatment the recombinant proteins were detected by flow cytometry using antibodies specific for Gaussia luciferase (GLuc) or the c-myc tag incorporated into the POI-FimH fusion proteins (Fig. 2A)
We describe a bacterial expression system using the bacterial fimbriae component FimH as a vehicle for bacterial surface display of various heterologous proteins while retaining their functional capacities
Summary
Bacterial surface display of recombinant proteins has become an attractive strategy for a broad range of applications such as production of bioadsorbents[1], generation of cellular biosensors[2], development of novel vaccine platforms[3], screening of antibody libraries[4] and whole-cell biocatalysis[5]. In Gram-negative bacteria the outer membrane generally acts as a barrier to restrict the protein export from the cell interior; only pilins, flagellins, specific surface enzymes, and a few bacterial toxins are transported across the outer membrane[16] These natural display systems have the benefit of being optimized for transporting and folding protein units to build polymeric structures on the extracellular surface making the display system attractive for biotechnological applications. Pallesen and colleagues used the positions 225 and 258 within the FimH adhesin to display the preS2 domain of the hepatitis B surface antigen or an epitope from cholera toxin[19] Both positions within the FimH protein proved to be suitable for the integration of peptides of up to 56 amino acids which could be produced, displayed on the cell surface and partially conserved the adhesive function of FimH19. Bacteria with surface displayed proteins can be used for screening purposes and, can be released in a functionally active form by specific proteolytic cleavage making the strategy attractive for protein production without the need to disrupt the bacteria by harsh procedures
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