Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification.
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 beta-galactosidase (lacZ), were successfully purified using the PhaR based protein purification method.


CONCLUSIONS
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.
Zhang
Shuang
S
Department of Biological Sciences and Biotechnology, School of Life Science, Tsinghua University, Beijing 100084, China.
Wang
Zhi Hui
ZH
Chen
Guo Qiang
GQ
D000345:Affinity Labels / Q000737:chemistry*; D003001:Cloning, Molecular / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D047668:Inteins / Q000235:genetics*; D053758:Nanoparticles; D054813:Polyhydroxyalkanoates / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D012097:Repressor Proteins / Q000737:chemistry*; D013696:Temperature
D000345:Affinity Labels; D029968:Escherichia coli Proteins; D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D012097:Repressor Proteins
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