Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

We present a one-step affinity purification set-up suitable for the purification of secreted proteins

J. Tykvart; P. Šácha; C. Bařinka; T. Knedlík; J. Starková; J. Lubkowski; J. Konvalinka

2011

Scholarcy highlights

  • Purification of recombinant proteins via diverse affinity tags has recently largely replaced approaches utilizing the biophysical features of the proteins
  • The DNA encoding AviTEV tag was prepared from oligonucleotides by the gene fusion approach and inserted into the plasmid pMNAEXST, which enables inducible expression in Drosophila Schneider 2 cells
  • In order to analyze the efficiency of Avi-GCPII in vivo biotinylation, three different plasmids were prepared encoding biotin-protein ligase from E. coli localized in cytoplasm, within the endoplasmic reticulum, and secreted into the media
  • Efficient, and robust recombinant protein expression and purification is indispensable for many areas of molecular biology, from structural genomics to proteinprotein interaction studies
  • We aimed to develop a well-functioning one-step purification procedure utilizing a biotin acceptor peptide as a purification tag, for its well-characterized properties of binding to streptavidin and its analogues, and Drosophila S2 cells as an expression system, for their relatively easy handling, possibility of large-scale growth in suspension, and high expression yields
  • We decided to locate the AviTEV-tag on the Nterminus of rhGCPII since its 3D structure indicates that it is more flexible than C-terminus of the molecule
  • Glutamate carboxypeptidase II extracellular portion of GCPII consisting of amino acids 44–750 molecule of rhGCPII fused at its N-terminus with AviTEV tag Tobacco Etch Virus protease N-acetyl-L-aspartyl-L-glutamate N-acetyl-L-aspartyl-L-methionine 2-(phosphonomethyl)-pentanedioic acid biotin acceptor peptide biotin-protein ligase from E. coli endoplasmic reticulum

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