An Evolved Mxe GyrA Intein for Enhanced Production of Fusion Proteins

Abstract

Expressing antibodies as fusions to the non-self-cleaving Mxe GyrA intein enables site-specific, carboxy-terminal chemical modification of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein fusion protein expression platforms typically yield insoluble inclusion bodies that require refolding to obtain active antibody-intein fusion proteins. Previously, we demonstrated that it was possible to employ yeast surface display to express properly folded single-chain antibody (scFv)-intein fusions, therefore permitting the direct small-scale chemical functionalization of scFvs. Here, directed evolution of the Mxe GyrA intein was performed to improve both the display and secretion levels of scFv-intein fusion proteins from yeast. The engineered intein was shown to increase the yeast display levels of eight different scFvs by up to 3-fold. Additionally, scFv- and green fluorescent protein (GFP)-intein fusion proteins can be secreted from yeast, and while fusion of the scFvs to the wild-type intein resulted in low expression levels, the engineered intein increased scFv-intein production levels by up to 30-fold. The secreted scFv- and GFP-intein fusion proteins retained their respective binding and fluorescent activities, and upon intein release, EPL resulted in carboxy-terminal azide functionalization of the target proteins. The azide-functionalized scFvs and GFP were subsequently employed in a copper-free, strain-promoted click reaction to site-specifically immobilize the proteins on surfaces, and it was demonstrated that the functionalized, immobilized scFvs retained their antigen binding specificity. Taken together, the evolved yeast intein platform provides a robust alternative to bacterial intein expression systems.

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

“…The protein‐intein fusion expression vector has been previously described . In this system, an anti‐fluorescein scFv (4‐4‐20) was used as the fusion partner to the engineered 202‐08 intein.…”

“…Saccharomyces cerevisiae strain YVH10 (MATα PDI1::GAPDHPDI1::LEU2 ura3−52 trp1 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL) was used for protein secretion. The engineered Mxe GyrA intein‐fused pRS316‐FLAG‐202‐08 vector was used as a backbone for protein secretion and plasmid mutagenesis, with the original vector having Gly preceding the intein (at the −1 position). 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.…”

“…The ability to release the scFv and GFP from secreted intein fusions was performed as described previously . Briefly, 1 M mercaptoethanesulfonic acid (MESNA, Sigma‐Aldrich) was added to scFv‐ or GFP‐containing supernatants or purified protein to a final concentration of 100 mM, and the reaction was allowed to proceed for 20 h at RT prior to anti‐FLAG Western blot analysis.…”

“…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. To remedy the deleterious effects of the intein fusion, an evolved Mxe GyrA intein named 202‐08 was developed that enhanced the secretion levels of scFv‐intein fusions to those of the unfused protein, and the resultant protein could be functionalized for downstream applications …”

“…Since large quantities of functionalized protein are often desirable for a variety of applications, we sought to identify approaches to further elevate the yield of EPL‐compatible proteins by making use of the 202‐08 intein. One approach is to increase the intein cleavage efficiency as previous work indicated incomplete cleavage of Mxe GyrA fusion proteins . Studies using bacterial production platforms have demonstrated that Mxe GyrA intein cleavage can be modulated by altering conditions such as pH and temperature .…”