In Vivo Site-Specific Protein Tagging with Diverse Amines Using an Engineered Sortase Variant.
Chemoenzymatic modification of proteins is an attractive option to create highly specific conjugates for therapeutics, diagnostics, or materials under gentle biological conditions. However, these methods often suffer from expensive specialized substrates, bulky fusion tags, low yields, and extra purification steps to achieve the desired conjugate. Staphylococcus aureus sortase A and its engineered variants are used to attach oligoglycine derivatives to the C-terminus of proteins expressed with a minimal LPXTG tag. This strategy has been used extensively for bioconjugation in vitro and for protein-protein conjugation in living cells. Here we show that an enzyme variant recently engineered for higher activity on oligoglycine has promiscuous activity that allows proteins to be tagged using a diverse array of small, commercially available amines, including several bioorthogonal functional groups. This technique can also be carried out in living Escherichia coli, enabling simple, inexpensive production of chemically functionalized proteins with no additional purification steps.
National Institute of Standards and Technology , Stanford, California 94305, United States.
Jeff E
JE
Glasgow
National Institute of Standards and Technology , Stanford, California 94305, United States.
Marc L
ML
Salit
Departments of Bioengineering and Chemical Engineering, Stanford University , Stanford, California 94305, United States.
Jennifer R
JR
Cochran
D000588:Amines / Q000737:chemistry*; D019881:Aminoacyltransferases / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000737:chemistry*; D015394:Molecular Structure; D015202:Protein Engineering / Q000379:methods*; D013211:Staphylococcus aureus / Q000201:enzymology*
D000588:Amines; D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D005998:Glycine