Cell surface display of CAL-B in Escherichia coli using the split GFP system

Abstract

Bacterial surface display systems enable the immobilization of proteins on the outer membrane for applications such as peptide library screening, biosensing, and bioadsorption. However, the display efficiency of large proteins remains limited, primarily due to challenges in translocating bulky polypeptides across the membrane. To address this, a split Green Fluorescent Protein (split GFP)-based strategy was evaluated. This method employs two non-fluorescent GFP fragments—GFP11 and GFP1–10—that reassemble into a fluorescent complex when co-localized. In this study, the small GFP fragment (GFP11) was genetically fused to Lipoprotein-Outer Membrane Protein A (Lpp-OmpA) to promote membrane anchoring, while the large GFP fragment (GFP1–10) was fused to Candida antarctica lipase B (CAL-B). The CAL-B-GFP1–10 fusion protein was expressed separately and then incubated with cells displaying Lpp-OmpA-GFP11, facilitating potential reassembly on the cell surface. Restoration of fluorescence served as an indirect indicator of successful surface localization. Enzymatic assays were also performed to compare the activity of CAL-B displayed via the split GFP system versus conventional direct fusion to Lpp-OmpA. The results demonstrated that the split GFP approach can enhance surface display and preserve enzymatic function, offering a promising alternative for displaying large or structurally complex proteins. While further optimization is needed, these findings support the potential of split GFP-assisted strategies in expanding the scope of bacterial surface display applications.