Activity-independent screening of secreted proteins using split GFP

Abstract

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 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.

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis

“…It contains the strong promoter P HpaII [ 22 ], a strong SD sequence (TAAGGAGG), and the start codon AUG previously described as being most efficient for protein production [ 12 ]. This vector and its derivatives were successfully used in several studies for the production and secretion of recombinant proteins in B. subtilis [ 4 , 5 , 31 , 36 , 41 , 42 ]. However, pBSMul1 contains a 4nt spacer (ACAT, Fig.…”

“…Both proteins were fused in-frame with the B. subtilis signal peptides of the extracellular protease Epr (SPEpr), the pectate lyase Pel (SPPel) and the extracellular ribonuclease Bsn (SPBsn). Signal peptides of Epr and Pel performed well in previous cutinase secretion screenings [ 4 ] whereas the signal peptides of Epr and Bsn improved EXLX1 secretion [ 31 ]. Both proteins were fused to a C-terminal split GFP tag (GFP11) allowing activity-independent quantification of Cut-11 and EXLX1-11 in vitro [ 31 ].…”

“…The GUS gene ( uidA ) was amplified from E. coli DH5α genomic DNA. Fusions of EXLX1 - 11 and cut - 11 , respectively, with the signal peptide sequences SPepr , SPpel and SPbsn were amplified from a previously constructed signal peptide library [ 31 ]. All gene fragments were ligated into the Nde I/ Xba I hydrolyzed E. coli – B. subtilis shuttle vector pBSMul1 [ 22 ].…”

“…The amount of secreted cutinase Cut-11 and swollenin EXLX1-11 was detected by the split GFP assay. The truncated GFP1-10 fragment (detector) was produced by E. coli BL21(DE3) with pET22- sfGFP1 - 10 as described previously [ 31 ]. 20 µl culture supernatant was mixed with 180 µl detector solution and incubated at room temperature for at least 16 h. Fluorescence was measured using the Tecan Infinite M1000 Pro microplate reader (Tecan, Männedorf, Switzerland).…”

Agar plate‐based screening methods for the identification of polyester hydrolysis by Pseudomonas species

“…Examples include transcriptional fusions confirming the successful transcription of target operons to identify promising expression strains (Domröse et al ., ), or as a model protein for studies on protein secretion, e.g. using signal peptide libraries (Knapp et al ., ). In addition, both substrates generally expand the set of polymers applicable to screenings.…”