Abstract
BackgroundThe split GFP assay is a well-known technology for activity-independent screening of target proteins. A superfolder GFP is split into two non-fluorescent parts, GFP11 which is fused to the target protein and GFP1-10. In the presence of both, GFP1-10 and the GFP11-tag are self-assembled and a functional chromophore is formed. However, it relies on the availability and quality of GFP1-10 detector protein to develop fluorescence by assembly with the GFP11-tag connected to the target protein. GFP1-10 detector protein is often produced in small scale shake flask cultivation and purified from inclusion bodies.ResultsThe production of GFP1-10 in inclusion bodies and purification was comprehensively studied based on Escherichia coli as host. Cultivation in complex and defined medium as well as different feed strategies were tested in laboratory-scale bioreactor cultivation and a standardized process was developed providing high quantity of GFP1-10 detector protein with suitable quality. Split GFP assay was standardized to obtain robust and reliable assay results from cutinase secretion strains of Corynebacterium glutamicum with Bacillus subtilis Sec signal peptides NprE and Pel. Influencing factors from environmental conditions, such as pH and temperature were thoroughly investigated.ConclusionsGFP1-10 detector protein production could be successfully scaled from shake flask to laboratory scale bioreactor. A single run yielded sufficient material for up to 385 96-well plate screening runs. The application study with cutinase secretory strains showed very high correlation between measured cutinase activity to split GFP fluorescence signal proofing applicability for larger screening studies.
Highlights
The split GFP assay is a well-known technology for activity-independent screening of target proteins
After purification of GFP1-10 we demonstrate the application of the detector solution to determine heterologous secretion of Fusarium solani f. sp. pisi cutinase with Corynebacterium glutamicum
Scaling from flask to laboratory‐scale bioreactor Inclusion body-based production of GFP1-10 with E. coli BL21(DE3) pET22b-sfGFP1-10 was done in 50 ml lysogeny broth (LB) in shake flasks and in 1 l LB in stirred tank reactors as batch processes
Summary
The split GFP assay is a well-known technology for activity-independent screening of target proteins. In the presence of both, GFP1-10 and the GFP11-tag are self-assembled and a functional chromophore is formed It relies on the availability and quality of GFP1-10 detector protein to develop fluorescence by assembly with the GFP11-tag connected to the target protein. In contrast to full length reporter proteins, only the 11th β-sheet of a superfolder GFP is used as a tag for detection. Since the β-sheet consists of only 16 amino acids connected to the target protein by a small peptide linker, the impact of the tag on solubility and folding of Müller et al Microb Cell Fact (2021) 20:191. The system was optimized for faster fluorescence formation by prematuration of the GFP110 [5, 6]. This could drastically reduce incubation times but requires additional purification steps. A broad review of recent developments and applications of the split GFP assay is provided by Pedelacq and Cabantous [9]
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