title: Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein.
abstract: BACKGROUND
  Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling.
  
  
  METHODS
  A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein.
  
  
  CONCLUSIONS
  We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.
authors.affiliation: Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Volkmann
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D047668:Inteins; D058619:Maltose-Binding Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D020040:Trans-Splicing
chemical_list: D005456:Fluorescent Dyes; D055767:Immobilized Proteins; D058619:Maltose-Binding Proteins; D010455:Peptides; D033902:Periplasmic Binding Proteins; D011506:Proteins; D011990:Receptors, Transferrin