title: Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.
abstract: We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.
authors.affiliation: Department of Chemistry, State University of New York at Binghamton, Binghamton, NY 13902, USA.
authors.forename: Jenny
authors.initials: J
authors.lastname: Tse
authors.affiliation: Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902, USA.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, State University of New York at Binghamton, Binghamton, NY 13902, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Zengeya
authors.affiliation: Department of Chemistry, State University of New York at Binghamton, Binghamton, NY 13902, USA.
authors.forename: Eriks
authors.initials: E
authors.lastname: Rozners
authors.affiliation: Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902, USA. Electronic address: annatan@binghamton.edu.
authors.forename: Anna
authors.initials: A
authors.lastname: Tan-Wilson
mesh_terms: D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005591:Chemical Fractionation / Q000379:methods*; D006801:Humans; D007461:Iodoacetates / Q000737:chemistry; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D007461:Iodoacetates; D020135:Peptide Nucleic Acids; D001710:Biotin; D019809:Streptavidin
keywords: Biotin–streptavidin technology; Iodoacetate; Peptide nucleic acid; Protein affinity purification; Proteomics; Thioreductase