Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

Gilead Sciences and IOCB Research Centre Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo n 2 Prague 6 Czech Republic

See more in PubMed

Green NM. Avidin. 1. The Use of (14-C)Biotin for Kinetic Studies and for Assay. Biochem J. 1963;89:585–591. PubMed PMC

Qureshi MH, Wong SL. Design, production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins. Protein Expr Purif. 2002;25:409–415. PubMed

Wu SC, Wong SL. Engineering soluble monomeric streptavidin with reversible biotin binding capability. J Biol Chem. 2005;280:23225–23231. PubMed

Lane MD, Rominger KL, Young DL, Lynen F. The Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and (+)-Biotin. Ii. Investigation of the Reaction Mechanism. J Biol Chem. 1964;239:2865–2871. PubMed

Lane MD, Young DL, Lynen F. The Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and (+)-Biotin. I. Purification of the Apoenzyme and Synthetase; Characteristics of the Reaction. J Biol Chem. 1964;239:2858–2864. PubMed

Beckett D, Kovaleva E, Schatz PJ. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 1999;8:921–929. PubMed PMC

Schatz PJ. Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology (N Y) 1993;11:1138–1143. PubMed

de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E, Heck A, Grosveld F, Strouboulis J. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A. 2003;100:7480–7485. PubMed PMC

Duffy S, Tsao KL, Waugh DS. Site-specific, enzymatic biotinylation of recombinant proteins in Spodoptera frugiperda cells using biotin acceptor peptides. Anal Biochem. 1998;262:122–128. PubMed

Predonzani A, Arnoldi F, Lopez-Requena A, Burrone OR. In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system. BMC Biotechnol. 2008;8:41. PubMed PMC

Barat B, Wu AM. Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum. Biomol Eng. 2007;24:283–291. PubMed PMC

Driegen S, Ferreira R, van Zon A, Strouboulis J, Jaegle M, Grosveld F, Philipsen S, Meijer D. A generic tool for biotinylation of tagged proteins in transgenic mice. Transgenic Res. 2005;14:477–482. PubMed

Sung K, Maloney MT, Yang J, Wu C. A novel method for producing monobiotinylated, biologically active neurotrophic factors: An essential reagent for single molecule study of axonal transport. J Neurosci Methods. 2011 PubMed PMC

Juzumiene D, Chang CY, Fan D, Hartney T, Norris JD, McDonnell DP. Single-step purification of full-length human androgen receptor. Nucl Recept Signal. 2005;3:e001. PubMed PMC

Yang J, Jaramillo A, Shi R, Kwok WW, Mohanakumar T. In vivo biotinylation of the major histocompatibility complex (MHC) class II/peptide complex by coexpression of BirA enzyme for the generation of MHC class II/tetramers. Hum Immunol. 2004;65:692–699. PubMed

Schmidt TG, Skerra A. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc. 2007;2:1528–1535. PubMed

Skerra A, Schmidt TG. Applications of a peptide ligand for streptavidin: the Strep-tag. Biomol Eng. 1999;16:79–86. PubMed

Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S. Comparison of affinity tags for protein purification. Protein Expr Purif. 2005;41:98–105. PubMed

Barinka C, Rinnova M, Sacha P, Rojas C, Majer P, Slusher BS, Konvalinka J. Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II. J Neurochem. 2002;80:477–487. PubMed

Barinka C, Sacha P, Sklenar J, Man P, Bezouska K, Slusher BS, Konvalinka J. Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity. Protein Sci. 2004;13:1627–1635. PubMed PMC

Buhler P, Wolf P, Elsasser-Beile U. Targeting the prostate-specific membrane antigen for prostate cancer therapy. Immunotherapy. 2009;1:471–481. PubMed

Elgamal AA, Holmes EH, Su SL, Tino WT, Simmons SJ, Peterson M, Greene TG, Boynton AL, Murphy GP. Prostate-specific membrane antigen (PSMA): current benefits and future value. Semin Surg Oncol. 2000;18:10–16. PubMed

Ghosh A, Heston WD. Tumor target prostate specific membrane antigen (PSMA) and its regulation in prostate cancer. J Cell Biochem. 2004;91:528–539. PubMed

Neale JH, Olszewski RT, Gehl LM, Wroblewska B, Bzdega T. The neurotransmitter N-acetylaspartylglutamate in models of pain, ALS, diabetic neuropathy, CNS injury and schizophrenia. Trends Pharmacol Sci. 2005;26:477–484. PubMed

Conway RE, Petrovic N, Li Z, Heston W, Wu D, Shapiro LH. Prostate-specific membrane antigen regulates angiogenesis by modulating integrin signal transduction. Mol Cell Biol. 2006;26:5310–5324. PubMed PMC

Liu T, Jabbes M, Nedrow-Byers JR, Wu LY, Bryan JN, Berkman CE. Detection of prostate-specific membrane antigen on HUVECs in response to breast tumor-conditioned medium. Int J Oncol. 2011;38:1349–1355. PubMed

Hlouchova K, Barinka C, Klusak V, Sacha P, Mlcochova P, Majer P, Rulisek L, Konvalinka J. Biochemical characterization of human glutamate carboxypeptidase III. J Neurochem. 2007;101:682–696. PubMed

Robinson MB, Blakely RD, Couto R, Coyle JT. Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain. J Biol Chem. 1987;262:14498–14506. PubMed

Rasband WS. ImageJ. U. S. National Institutes of Health. Bethesda, Maryland, USA: 1997–2008. http://rsb.info.nih.gov/ij/

Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Method Enzymol. 1997;276:307–326. PubMed

Barinka C, Rovenska M, Mlcochova P, Hlouchova K, Plechanovova A, Majer P, Tsukamoto T, Slusher BS, Konvalinka J, Lubkowski J. Structural insight into the pharmacophore pocket of human glutamate carboxypeptidase II. J Med Chem. 2007;50:3267–3273. PubMed

Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53:240–255. PubMed

Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60:2126–2132. PubMed

Chen VB, Arendall WB, 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010;66:12–21. PubMed PMC

Leatherbarrow RJ. GraFit Version 5. Horley, UK: Erithacus Software Limited; 2001.

Woodward C, Henderson JK, Wielgos T. High-speed amino acid analysis (AAA) on 1.8 µm reversed-phase (RP) columns. Agilent Technologies App. Note. 2007:5989–6297. EN.

Sacha P, Zamecnik J, Barinka C, Hlouchova K, Vicha A, Mlcochova P, Hilgert I, Eckschlager T, Konvalinka J. Expression of glutamate carboxypeptidase II in human brain. Neuroscience. 2007;144:1361–1372. PubMed

Barinka C, Starkova J, Konvalinka J, Lubkowski J. A high-resolution structure of ligand-free human glutamate carboxypeptidase II. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007;63:150–153. PubMed PMC

Jackson PF, Cole DC, Slusher BS, Stetz SL, Ross LE, Donzanti BA, Trainor DA. Design, synthesis, and biological activity of a potent inhibitor of the neuropeptidase N-acetylated alpha-linked acidic dipeptidase. J Med Chem. 1996;39:619–622. PubMed

Phan J, Zdanov A, Evdokimov AG, Tropea JE, Peters HK, 3rd, Kapust RB, Li M, Wlodawer A, Waugh DS. Structural basis for the substrate specificity of tobacco etch virus protease. J Biol Chem. 2002;277:50564–50572. PubMed

Mlcochova P, Plechanovova A, Barinka C, Mahadevan D, Saldanha JW, Rulisek L, Konvalinka J. Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis. FEBS J. 2007;274:4731–4741. PubMed

Bucher MH, Evdokimov AG, Waugh DS. Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein. Acta Crystallogr D Biol Crystallogr. 2002;58:392–397. PubMed

Schrodinger, LLC, The PyMOL Molecular Graphics System. Version 1.3r1. 2010

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