Activity-independent screening of secreted proteins using split GFP

Andreas Knapp, Myriam Ripphahn, Kristina Volkenborn, Pia Skoczinski, Karl-Erich Jaeger
2017 Journal of Biotechnology  
A B S T R A C T The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic
more » ... on of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner. http://dx.
doi:10.1016/j.jbiotec.2017.05.024 pmid:28619616 fatcat:4b5ft7bkdvdgtdskwlotxitawi