Facile chemical functionalization of proteins through intein-linked yeast display.
Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we have extended these methods to release and modify intein fusion proteins expressed on the yeast surface, thereby eliminating the need for soluble protein expression and purification. To this end, we sought to simultaneously release yeast surface-displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry. Single-chain antibodies (scFv) and green fluorescent protein (GFP) were displayed on the yeast surface as fusions to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released from the yeast surface with either a sulfur nucleophile (MESNA) or a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine azide permitted the simultaneous release and azido functionalization of displayed proteins, but nonspecific reactions with other yeast proteins were detected, and cleavage efficiency was limited. In contrast, MESNA released significantly more protein from the yeast surface while also generating a unique thioester at the carboxy-terminus of the released protein. These protein thioesters were subsequently reacted with a cysteine alkyne in an EPL reaction and then employed in an azide-alkyne cycloaddition to immobilize the scFv and GFP on an azide-decorated surface with >90% site-specificity. Importantly, the immobilized proteins retained their activity. Since yeast surface display is also a protein engineering platform, these approaches provide a particularly powerful tool for the rapid assessment of engineered proteins.
Department of Chemical and Biological Engineering , University of Wisconsin-Madison, 1415 Engineering Drive, Madison, Wisconsin 53706, United States.
Carrie J
CJ
Marshall
Nitin
N
Agarwal
Jeet
J
Kalia
Vanessa A
VA
Grosskopf
Nicholas A
NA
McGrath
Nicholas L
NL
Abbott
Ronald T
RT
Raines
Eric V
EV
Shusta
D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D057930:Click Chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D055767:Immobilized Proteins / Q000737:chemistry* / Q000235:genetics; D047668:Inteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics
D000480:Alkynes; D001386:Azides; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins