Functional Cell-Surface Display of a Lipase-Specific Chaperone

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated “Lif” proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell‐surface display of the Lif chaperone and FACS (fluorescence‐activated cell sorting)‐based analysis of bacterial cells that carried foldase–lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh‐throughput screening of large libraries of foldase variants generated by directed evolution. Lifs like these. Several bacterial lipases of biotechnological importance need specific chaperones, termed lipase specific foldases (Lifs), to fold into an enzymatically active conformation. Here, the functional cell‐surface display of the Lif protein from Pseudomonas aeruginosa is demonstrated (see figure). This foldase autodisplay system could enable the study of protein–protein interactions and allow the ultrahigh‐throughput screening of large libraries of foldase variants.