title: Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification.
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 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.
authors.affiliation: Department of Biological Sciences and Biotechnology, School of Life Science, Tsinghua University, Beijing 100084, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Zhang
authors.forename: Zhi Hui
authors.initials: ZH
authors.lastname: Wang
authors.forename: Guo Qiang
authors.initials: GQ
authors.lastname: Chen
mesh_terms: 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
chemical_list: D000345:Affinity Labels; D029968:Escherichia coli Proteins; D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D012097:Repressor Proteins
references.citation: Nucleic Acids Res. 1998 Nov 15;26(22):5109-15
references.pmid: 9801307
references.citation: Biotechnol Prog. 1992 Jul-Aug;8(4):347-52
references.pmid: 1368456
references.citation: EMBO J. 1998 Feb 16;17(4):918-26
references.pmid: 9463370
references.citation: Nat Biotechnol. 1999 Nov;17(11):1112-5
references.pmid: 10545920
references.citation: Biotechniques. 1999 Jul;27(1):110-4, 116, 118-20
references.pmid: 10407673
references.citation: J Biol Chem. 2004 Aug 20;279(34):35281-6
references.pmid: 15194682
references.citation: J Biol Chem. 1999 Jun 25;274(26):18359-63
references.pmid: 10373440
references.citation: Lab Chip. 2008 Nov;8(11):1957-62
references.pmid: 18941699
references.citation: Protein Sci. 2005 Jun;14(6):1387-95
references.pmid: 15883185
references.citation: Biotechnol J. 2006 Feb;1(2):113
references.pmid: 16892236
references.citation: Gene. 1999 Apr 29;231(1-2):1-13
references.pmid: 10231563
references.citation: J Bacteriol. 2007 Feb;189(3):1118-27
references.pmid: 17122335
references.citation: Biotechnol Prog. 2000 Nov-Dec;16(6):1055-63
references.pmid: 11101334
references.citation: Protein Expr Purif. 2009 Jun;65(2):261-6
references.pmid: 19297701
references.citation: Methods Mol Biol. 2005;310:123-30
references.pmid: 16350951
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Biomacromolecules. 2006 Aug;7(8):2449-54
references.pmid: 16903695
references.citation: Methods. 2001 Jul;24(3):257-77
references.pmid: 11403575
references.citation: Protein Sci. 1998 Nov;7(11):2256-64
references.pmid: 9827992
references.citation: Chem Rec. 2006;6(4):183-93
references.pmid: 16900466
references.citation: Nat Methods. 2005 Sep;2(9):659-61
references.pmid: 16074986
references.citation: Methods Enzymol. 2000;326:376-418
references.pmid: 11036654
references.citation: Gene. 1997 Jun 19;192(2):271-81
references.pmid: 9224900
references.citation: Appl Microbiol Biotechnol. 2003 Jan;60(5):523-33
references.pmid: 12536251
references.citation: J Biotechnol. 2006 Aug 20;125(1):48-56
references.pmid: 16546284
references.citation: Gene. 1988 Jul 15;67(1):31-40
references.pmid: 3047011
references.citation: Protein Expr Purif. 2005 Sep;43(1):26-32
references.pmid: 15979896
references.citation: Nat Biotechnol. 2001 Oct;19(10):974-7
references.pmid: 11581666
references.citation: J Bacteriol. 2002 Jul;184(14):3992-4002
references.pmid: 12081972
references.citation: Protein Expr Purif. 2001 Jun;22(1):135-40
references.pmid: 11388811