In vivo biotinylated proteins as targets for phage-display selection experiments

https://doi.org/10.1016/j.pep.2004.05.012Get rights and content

Abstract

Screening phage-displayed combinatorial libraries represents an attractive method for identifying affinity reagents to target proteins. Two critical components of a successful selection experiment are having a pure target protein and its immobilization in a native conformation. To achieve both of these requirements in a single step, we have devised cytoplasmic expression vectors for expression of proteins that are tagged at the amino- or carboxy-terminus (pMCSG16 and 15) via the AviTag, which is biotinylated in vivo with concurrent expression of the BirA biotin ligase. To facilitate implementation in high-throughput applications, the engineered vectors, pMCSG15 and pMCSG16, also contain a ligase-independent cloning site (LIC), which permits up to 100% cloning efficiency. The expressed protein can be purified from bacterial cell lysates with immobilized metal affinity chromatography or streptavidin-coated magnetic beads, and the beads used directly to select phage from combinatorial libraries. From selections using the N-terminally biotinylated version of one target protein, a peptide ligand (Kd=9μM) was recovered that bound in a format-dependent manner. To demonstrate the utility of pMCSG16, a set of 192 open reading frames were cloned, and protein was expressed and immobilized for use in high-throughput selections of phage-display libraries.

Section snippets

Reagents

All plasmids were propagated and proteins were expressed in the E. coli strains XL1 Blue (Stratagene, La Jolla, CA) and BL21(DE3) (Novagen, Madison, WI), respectively. The pBirA plasmid was purchased from Avidity (Denver, CO) and E. coli XL1 Blue F tet host cells were from Stratagene (La Jolla, CA). Nickel–NTA agarose was purchased from Qiagen (Germantown, MD). Restriction endonucleases, T4 DNA polymerase, and Advantage cDNA polymerase were purchased from New England Biolabs (Beverly, MA),

Construction of pMCSG16 and pMCSG15

Two expression vectors were constructed to generate N- and C-terminal fusions for in vivo biotinylation. Vectors were generated by incorporating double-stranded oligonucleotides encoding the AviTag, a 15 amino acid long substrate for BirA, into the pMCSG7 plasmid [19]. The resulting recombinant, named pMCSG16 (Fig. 1A), encodes an N-terminal six histidine tag, AviTag, a (Gly–Ser)2 linker, the seven amino acid TEV protease cleavage site (ENLYFQS), and a LIC site. A second vector was constructed

Discussion

Biotin (vitamin H) is a small coenzyme that is synthesized by plants, most bacteria, and some fungi, and is primarily bound to protein in the cell. The BirA protein (biotin ligase) transfers biotin to the ε-amino group of specific lysine residue in an ATP-dependent process [29]. To define the substrate specificity of the BirA enzyme, Schatz [30] used phage display to find a 13-residue consensus sequence that is recognized and biotinylated by the enzyme. This sequence, and a 15-residue sequence

Acknowledgements

We acknowledge the editorial comments of Drs. John Kehoe, Ushma Kriplani, and Zhao Zhong Han. A special thanks to Dr. David Waugh (National Cancer Institute, Frederick, MD) for the TEV protease expression vector. This work was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract W-31-109-Eng-38.

References (50)

  • L. Dieckman et al.

    High throughput methods for gene cloning and expression

    Protein Expr. Purif.

    (2002)
  • L. Stols et al.

    A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site

    Protein Expr. Purif.

    (2002)
  • J.E. Butler et al.

    The immunochemistry of sandwich ELISAs–VI. Greater than 90% of monoclonal and 75% of polyclonal anti-fluorescyl capture antibodies (CAbs) are denatured by passive adsorption

    Mol. Immunol.

    (1993)
  • M. Yamabhai et al.

    Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains

    J. Biol. Chem.

    (1998)
  • A. Hemminki et al.

    Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs

    Immunotechnology

    (1998)
  • R.E. Hawkins et al.

    Selection of phage antibodies by binding affinity. Mimicking affinity maturation

    J. Mol. Biol.

    (1992)
  • F. D'Mello et al.

    An improved selection procedure for the screening of phage display peptide libraries

    J. Immunol. Methods

    (2001)
  • G.P. Smith et al.

    Libraries of peptides and proteins displayed on filamentous phage

    Methods Enzymol.

    (1993)
  • A. Chapman-Smith et al.

    The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity

    Trends Biochem Sci.

    (1999)
  • J.E. Cronan

    Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins

    J. Biol. Chem.

    (1990)
  • D. Samols et al.

    Evolutionary conservation among biotin enzymes

    J. Biol. Chem.

    (1988)
  • S. Duffy et al.

    Site-specific, enzymatic biotinylation of recombinant proteins in Spodoptera frugiperda cells using biotin acceptor peptides

    Anal. Biochem.

    (1998)
  • S. Athavankar et al.

    Control of gene expression with small molecules: biotin-mediated acylation of targeted lysine residues in recombinant yeast

    Chem. Biol.

    (2003)
  • A. Viens et al.

    Use of protein biotinylation in vivo for chromatin immunoprecipitation

    Anal. Biochem.

    (2004)
  • W.G. Dougherty et al.

    Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase

    Virology

    (1989)
  • Cited by (43)

    • Advances in antibody phage display technology

      2022, Drug Discovery Today
      Citation Excerpt :

      The AviTag sequence is biotinylated at its lysine residue by the Escherichia coli biotin ligase, BirA.33 The AviTagged antigen can be co-expressed with BirA in bacterial cells, yeast, and mammalian cells to achieve in vivo biotinylation.34,35 Alternatively, purified AviTagged antigen can be incubated with purified BirA and biotin to achieve in vitro biotinylation.36

    • A knottin scaffold directs the CXC-chemokine-binding specificity of tick evasins

      2019, Journal of Biological Chemistry
      Citation Excerpt :

      Following signal peptide cleavage, the expected sequence at the N terminus is DGG, predicted by SignalP 3.0 (44). For bacterial expression plasmids, parts included a modified E. coli_lacI:T7:lacO:RBS promoter (from pET28, Novagen), a His12-SUMO tag, an Avitag (45), and E. coli birA (from Addgene 20856, pDisplay-BirA-ER). The mature peptide encoding sequences of CXCL1 (Ala35–Asn107), CXCL7 (Ala59–Asp128), and CXCL9 (Thr23–Thr125) were cloned in-frame following the SUMO tag such that the N terminus would be correctly created following SUMO protease cleavage.

    • Poloxamer 188 decreases membrane toxicity of mutant SOD1 and ameliorates pathology observed in SOD1 mouse model for ALS

      2018, Neurobiology of Disease
      Citation Excerpt :

      Male B6SJL-Tg(SOD1*G93A)1Gur/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Human wild-type (WT)-, A4V-, and G93A-SOD1 and a control protein, the Src homology 3 (SH3) domain of c-Src kinase, were expressed in E. coli BL21 (DE3), and purified on a Ni-column (Qiagen, Valencia, CA) (Allen et al., 2012; Scholle et al., 2004). Proteins were desalted using 9 K protein concentrators (Pierce, Rockford, IL) and stored in HBS (20 mM Hepes, 100 mM NaCl, pH 7.4) with 2.5% glycerol at −80 °C.

    View all citing articles on Scopus
    View full text