Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

Abstract

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.

Impacts of the −1 Amino Acid on Yeast Production of Protein‐Intein Fusions

“…The 202‐08 intein undergoes an N‐ to S‐acyl shift at its N‐terminal cysteine forming a thioester intermediate susceptible to nucleophilic attack. Upon reaction with the sulfur nucleophile MESNA, the protein, scFv 4‐4‐20, is cleaved from the intein, and a reactive thioester is formed at the C‐terminus of the released protein (Figure b) . The secreted product can then be assayed with an anti‐FLAG tag Western blot to identify the fraction of produced protein that has undergone successful cleavage.…”

“…Gly (−1) was mutated to 19 other amino acids by site‐directed mutagenesis (Q5 site‐directed mutagenesis kit, NEB) and primers designed by the Q5 NEB software. scFv2224, scFvH7, scFv4‐4‐20, and GFP were subcloned to appropriate pRS316‐FLAG‐202‐08 vectors from pCT4Re plasmids generated in a previous study …”

“…For glutathione (GSH)‐induced cleavage, 30 μM or 10 mM of reduced GSH was used for 20 h at room temperature at pH 6 in order to keep glutathione reduced and pH conditions similar to that of culture supernatants. To generate biotin functionalized proteins through EPL, 1 mM of a biotinylated cysteine peptide (BioP1 synthesized by the University of Wisconsin Biotechnology Center, Sequence: NH 2 ‐CDPEK(Bt)DS‐CONH 2 ) was added along with MESNA to buffer exchanged supernatants (Supporting Information Figure S1) or purified protein (Figure 6c), and the reaction was allowed to proceed for 20 h prior to anti‐biotin Western blot.…”

“…Subsequently, intein‐mediated protein splicing is activated by the addition of a thiol nucleophile that releases the target protein from the intein moiety while appending a reactive C‐terminal thioester intermediate . The thioester can subsequently react with an N‐terminal cysteine derivative to form a native amine bond enabling the addition of numerous chemical functionalities . Recently, we described using this approach to functionalize proteins produced in yeast; however, protein fusion to the wild‐type Mxe GyrA intein decreased production levels.…”

Cell surface engineering of industrial microorganisms for biorefining applications

“…A novel surface display using intein-based cell-surface system recently has been reported (Marshall et al, 2013). Using this system, several proteins, including xylose isomerase (Ota et al, 2013), laccase (Nakanishi et al, 2012), and expansin-like protein , have been displayed on the yeast cell surface.…”