Autodisplay made easy: a plasmid toolbox for the surface display of recombinant proteins and its optimization.

A plasmid library is provided for the surface display of proteins, to be used by experts and non experts, including a simple option for optimization. Based on a classical autotransporter protein, variants were constructed with different signal peptides, linkers and adjacent regions, 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 as model passenger was subjected to combinatorial permutation using the plasmids of the ATB (pATB). The best combination based on enzymatic activity was pATB_45-β-Gluc with an OprF signal peptide, a C terminal spacer (GGGDDNAAPA), and a short linker (ΔepitopeΔβ1). Activity was 4.9-fold increased compared to the initial construct. ATB can be extended to 72 variants by using two additional promoters (PRox306, ParaBAD) and to 81 variants by using an inverse autotransporter protein (YeeJ). Besides using the pATB library, individual plasmids can be picked for custom-made surface display.