Scaling production of GFP1-10 detector protein in E. coli for secretion screening by split GFP assay

Microbial Cell Factories

Chika L. Igwe¹  Wolfgang Wiechert²  Carolin Müller³  Marco Oldiges³ 

[1] Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany;Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany;Computational Systems Biotechnology (AVT.CSB), RWTH Aachen University, Aachen, Germany;Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany;Institute of Biotechnology, RWTH Aachen University, Aachen, Germany;

关键词: Escherichia coli;    Inclusion body;    Fed-batch;    Split GFP assay;    Corynebacterium glutamicum;    Signal peptide screening;    Cutinase;   

DOI  :  10.1186/s12934-021-01672-6

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY