NDLI: Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification

Content Provider	Springer Nature : BioMed Central
Author	Zhang, Shuang Wang, Zhi Hui Chen, Guo Qiang
Abstract	Background PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation. Results Fusion protein PhaR-intein-target protein was expressed in recombinant Escherichia coli. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target protein was released from the particles and purified after centrifugation. As target proteins, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and β-galactosidase (lacZ), were successfully purified using the PhaR based protein purification method. Conclusion The successful purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based in vitro purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple protein purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial protein purification systems.
Related Links	https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/1475-2859-9-28.pdf
Ending Page	6
Page Count	6
Starting Page	1
File Format	HTM / HTML
ISSN	14752859
DOI	10.1186/1475-2859-9-28
Journal	Microbial Cell Factories
Issue Number	1
Volume Number	9
Language	English
Publisher	BioMed Central
Publisher Date	2010-05-10
Access Restriction	Open
Subject Keyword	Applied Microbiology Biotechnology Microbiology Microbial Genetics and Genomics Enzymology Genetic Engineering Enhance Green Fluorescent Protein Protein Purification Maltose Binding Protein Crude Cell Lysate Express Protein Ligation
Content Type	Text
Resource Type	Article
Subject	Applied Microbiology and Biotechnology Bioengineering Biotechnology
Journal Impact Factor	4.3/2023
5-Year Journal Impact Factor	5.5/2023

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