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

Shuang Zhang,Zhi Hui Wang,Guo Qiang Chen

doi:10.1186/1475-2859-9-28

Shuang Zhang, Zhi Hui Wang + Show 1 more

Open Access

https://doi.org/10.1186/1475-2859-9-28

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Abstract

BackgroundPhaR 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.ResultsFusion 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.ConclusionThe 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.

Highlights

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
Three distinct bands of fusion proteins PhaR-intein-enhanced green fluorescent protein (EGFP) (RI:EGFP) (Fig. 3A, lane 2), PhaR-intein-maltose-binding protein (MBP) (RI:MBP) (Fig. 3B, lane 2) and PhaR-intein-LacZ (RI:LacZ) (Fig. 3C, lane 2) in soluble cell lysates were observed compared with pre-induced whole cell lysate (Fig. 3, lane 1)
After co-incubation of soluble cell lysates and PHBHHx nanoparticles, proteins attached on PHBHHx nanoparticles were shown in lane

Summary

Introduction

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 PhaRtagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. The target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were separated by centrifugation. A large amount of protein purification tags are available [3], such as polyhistidine (Poly-His), maltose-binding protein (MBP) and glutathione S-transferase (GST) [4,5,6]. The cost of affinity resins and other required reagents are the major obstacles that hinder these tags to be developed into large-scale protein production.

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