A New Bacterial Chassis for Enhanced Surface Display of Recombinant Proteins.

Abstract

Bacterial surface display is a valuable biotechnology technique for presenting proteins and molecules on the outer surface of bacterial cells. However, it has limitations, including potential toxicity to host bacteria and variability in display efficiency. To address these issues, we investigated the removal of abundant non-essential outer membrane proteins (OMPs) in E. coli as a new strategy to improve the surface display of recombinant proteins. We targeted OmpA, a highly prevalent OMP in E. coli, using the lambda red method. We successfully knocked out ompA in two E. coli strains, K-12 MG1655 and E. coli BL-21, which have broad research and therapeutic applications. We then combined ompA knockout strains and two OMPs with three therapeutic proteins including an anti-toxin enzyme (ClbS), interleukin 18 (IL-18) for activating cytotoxic T cells and an anti- CTLA4 nanobody (αCTLA4) for immune checkpoint blockade. A total of six different display constructs were tested for their display levels by flow cytometry, showing that the ompA knockout strains increased the percentage as well as the levels of display in bacteria compared to those of isogenic wild-type strains. By removing non-essential, highly abundant surface proteins, we develop an efficient platform for displaying enzymes and antibodies, with potential industrial and therapeutic applications. Additionally, the enhanced therapeutic efficacy opens possibilities for live bacteria-based therapeutics, expanding the technology's relevance in the field. The online version contains supplementary material available at 10.1007/s12195-024-00819-w.