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

Area of Science:

• Synthetic Biology
• Protein Engineering
• Biotechnology

Background:

• Bacterial surface display enables protein immobilization for various biotechnological applications.
• Current systems face limitations in displaying large or complex proteins due to translocation challenges.
• Split protein systems offer a potential solution by enabling protein assembly after translocation.

Purpose of the Study:

• To evaluate a split Green Fluorescent Protein (GFP) system for enhanced bacterial surface display of large proteins.
• To assess the functionality of a displayed enzyme using this novel system.
• To compare the split GFP approach with conventional direct fusion methods.

Main Methods:

• Genetically fused a small GFP fragment (GFP11) to a membrane anchor (Lpp-OmpA).
• Fused the large GFP fragment (GFP1-10) to Candida antarctica lipase B (CAL-B).
• Co-incubated cells displaying Lpp-OmpA-GFP11 with CAL-B-GFP1-10 and assessed surface localization via fluorescence and enzymatic activity.

Main Results:

• The split GFP system facilitated the surface display of CAL-B.
• Restored fluorescence indicated successful reassembly and localization of GFP fragments on the cell surface.
• Enzymatic assays showed preserved activity of CAL-B displayed via the split GFP system compared to direct fusion.

Conclusions:

• The split GFP strategy effectively enhances the surface display of large proteins like CAL-B.
• This approach preserves the enzymatic function of the displayed protein.
• Split GFP-assisted strategies offer a promising alternative for expanding bacterial surface display applications.