title: Site-specific chemical protein conjugation using genetically encoded aldehyde tags.
abstract: We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with α-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.
authors.affiliation: Redwood Bioscience Inc., Emeryville, California, USA.
authors.forename: David
authors.initials: D
authors.lastname: Rabuka
authors.forename: Jason S
authors.initials: JS
authors.lastname: Rush
authors.forename: Gregory W
authors.initials: GW
authors.lastname: deHart
authors.forename: Peng
authors.initials: P
authors.lastname: Wu
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D000447:Aldehydes / Q000737:chemistry*; D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D013058:Mass Spectrometry; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013429:Sulfatases / Q000378:metabolism
chemical_list: D000447:Aldehydes; D011506:Proteins; D011994:Recombinant Proteins; C524739:C(alpha)-formylglycine; D050862:Oxidoreductases Acting on Sulfur Group Donors; C480483:SUMF1 protein, human; D013429:Sulfatases; D003545:Cysteine; D000409:Alanine; D005998:Glycine