Autodisplay-Toolbox: How to Surface Display Proteins (Fast).

The display of proteins at the bacterial cell surface has appealing benefits for the analysis of binding partner, screening applications and whole cell biocatalysis. Here, a plasmid library is presented for the surface display of proteins, to be used by experts and non-experts, including a simple, but powerful option for optimization. Based on a classical (EhaA) autotransporter protein, variants were constructed with four different signal peptides (including adjacent region) and six different linkers. The variants were set under control of a rhaBAD promoter, resulting in a library of 24 plasmids named Autodisplay-ToolBox (ATB). For proof of concept, a β-glucosidase (β-Gluc) as model passenger was subjected to combinatorial permutation using the plasmids of the ATB (pATB) with subsequent pATB library screening. The best combination based on enzymatic activity was pATB_45-β-Gluc with an OprF signal peptide, an adjacent C-terminal spacer (GGGDDNAAPA), and a comparably short linker (ΔepitopeΔβ1). Its activity was 4.9-fold increased compared to the initial construct. The ATB can be extended by two promoters (P_Rox306, P_araBAD) and by variants of an inverse AT (YeeJ), to optimally control the expression strength and to enable inversed protein display, respectively. Besides using the whole pATB library, individual plasmids can be ordered from addgene for custom-made surface display.