Sortase A as a novel molecular "stapler" for sequence-specific protein conjugation.
The Sortase family of transpeptidase enzymes catalyzes sequence-specific ligation of proteins to the cell wall of Gram-positive bacteria. Here, we describe the application of recombinant Staphylococcus aureus Sortase A to attach a tagged model protein substrate (green fluorescent protein) to polystyrene beads chemically modified with either alkylamine or the in vivo Sortase A ligand, Gly-Gly-Gly, on their surfaces. Furthermore, we show that Sortase A can be used to sequence-specifically ligate eGFP to amino-terminated poly(ethylene glycol) and to generate protein oligomers and cyclized monomers using suitably tagged eGFP. We find that an alkylamine can substitute for the natural Gly3 substrate, which suggests the possibility of using the enzyme in materials applications. The highly specific and mild Sortase A-catalyzed reaction, based on small recognition tags unlikely to interfere with protein expression, thus represents a useful addition to the protein immobilization and modification tool kit.
Parthasarathy
Ranganath
R
Departments of Chemical and Biomolecular Engineering and Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Subramanian
Shyamsundar
S
Boder
Eric T
ET
D000588:Amines / Q000737:chemistry / Q000378:metabolism; D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002473:Cell Wall / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004925:Escherichia / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D011137:Polystyrenes / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology
D000588:Amines; D001426:Bacterial Proteins; D009842:Oligopeptides; D011137:Polystyrenes; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; C035647:glycyl-glycyl-glycine; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases