Expression of an Anti-GFP Nanobody on Escherichia coli UT5600 Using the BrkA Autotransporter System

Abstract

The BrkAutoDisplay system is optimized to display exogenous proteins on the surface of Escherichia coli cells, making it an attractive tool for the expression of recombinant proteins. The anti-GFP nanobody has been previously expressed in the IgA protease autotransporter while still retaining its original antigen-binding capacity, which is promising for further nanobody research. In this study, we investigated whether the anti-GFP nanobody, containing a disulfide bond, can be expressed using the BrkA autotransporter. To explore this, we engineered the recombinant pGFPNB construct by inserting the anti-GFP nanobody DNA sequence into the BrkA passenger domain through Gibson assembly. Western blot analysis confirmed that BrkA-GFPNB was expressed intracellularly in E. coli UT5600 cells. This study expands our knowledge of the exogenous proteins that can be expressed using the BrkA autotransporter as we demonstrated that the BrkA-GFPNB protein can be expressed. Further study is necessary in determining whether this protein of interest can be exported to bacterial surfaces and whether it retains its functionality.