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

Abstract

Expressing antibodies as fusions to the non‐self‐cleaving Mxe GyrA intein allows for site‐specific chemical functionalization via expressed protein ligation. It is highly desirable to maximize the yield of functionalizable protein; and previously an evolved intein, 202‐08, was identified that could increase protein fusion production in yeast. Given that the −1 amino acid residue upstream of inteins can affect cleavage efficiency, we examined the effects of amino acid variability at this position on 202‐08 intein cleavage efficiency and secretion yield. Varying the −1 residue resulted in a wide range of cleavage behaviors with some amino acids yielding substantial autocleaved product that could not be functionalized. Autocleavage was noticeably higher with the 202‐08 intein compared with the wild‐type Mxe GyrA intein and resulted directly from the catalytic activity of the intein. Refeeding of production cultures with nitrogen base and casamino acids reduced, but did not eliminate autocleavage, while increasing protein‐intein production up to seven‐fold. Importantly, two amino acids, Gly and Ala, at the −1 position resulted in good cleavage efficiency with no undesirable autocleavage, and can be used in concert with refeeding strategies to increase total functionalizable protein yield for multiple protein fusion partners. Taken together, we describe an optimized yeast expression platform for protein‐intein fusions. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2736, 2019

TSGIT: An N‐ and C‐terminal tandem tag system for purification of native and intein‐mediated ligation‐ready proteins

“…Additionally, SDS‐PAGE showed that the ~35% of unbound biotin‐labeled mRuby3 was generated by an incomplete IPL reaction. The IPL reaction and Intein cleavage have been previously shown to be strongly dependent on the C‐terminal amino acid of the protein of interest 32,42 . Moreover, the efficiency of cleavage and IPL were shown to be able to increase with longer (up to 40 hr) incubation times.…”

“…The IPL reaction and Intein cleavage have been previously shown to be strongly dependent on the C-terminal amino acid of the protein of interest. 32,42 Moreover, the efficiency of cleavage and IPL were shown to be able to increase with longer (up to 40 hr) incubation times. Nevertheless, we intentionally restricted the IPL reaction time to only 12 h to capture this limitation.…”