title: In vivo polyester immobilized sortase for tagless protein purification.
abstract: BACKGROUND
  Laboratory scale recombinant protein production and purification techniques are often complicated, involving multiple chromatography steps and specialized equipment and reagents. Here it was demonstrated that recombinant proteins can be expressed as covalently immobilized to the surface of polyester (polyhydroxyalkanoate, PHA) beads in vivo in Escherichia coli by genetically fusing them to a polyester synthase gene (phaC). The insertion of a self-cleaving module, a modified sortase A (SrtA) from Staphylococcus aureus and its five amino acid recognition sequence between the synthase and the target protein led to a simple protein production and purification method.
  
  
  RESULTS
  The generation of hybrid genes encoding tripartite PhaC-SrtA-Target fusion proteins, enabled immobilization of proteins of interest to the surface of PHA beads in vivo. After simple cell lysis and isolation of the PHA beads, the target proteins could be selectively and efficiently released form the beads by activating the sortase with CaCl2 and triglycine. Up to 6 mg/l of soluble proteins at a purity of ~98 % could be isolated in one step with no optimization. This process was used to produce and isolate three proteins: Green fluorescent protein, maltose binding protein and the Mycobacterium tuberculosis vaccine candidate Rv1626.
  
  
  CONCLUSIONS
  We have developed a new technique for easy production and purification of recombinant proteins. This technique is capable of producing and purifying high yields of proteins suitable for research application in less than 2 days. No costly or specialized protein chromatography equipment, resins, reagents or expertise are required.
authors.affiliation: Department of Microbiology, Monash University, Clayton, 3800, Australia. iain.hay@monash.edu.|Polybatics Ltd., Palmerston North, New Zealand. iain.hay@monash.edu.
authors.forename: Iain D
authors.initials: ID
authors.lastname: Hay
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand. j.du@massey.ac.nz.
authors.forename: Jinping
authors.initials: J
authors.lastname: Du
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand. P.RubioReyes@massey.ac.nz.
authors.forename: Patricia Rubio
authors.initials: PR
authors.lastname: Reyes
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand. b.rehm@massey.ac.nz.|MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington, New Zealand. b.rehm@massey.ac.nz.|Polybatics Ltd., Palmerston North, New Zealand. b.rehm@massey.ac.nz.
authors.forename: Bernd H A
authors.initials: BH
authors.lastname: Rehm
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011091:Polyesters / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D011091:Polyesters; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C035647:glycyl-glycyl-glycine; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D002122:Calcium Chloride