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
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Nov 23, 2018
- Source ID
- 10.1002/btpr.2736
Entities
People
- Eric V Shusta
- Loukas I. Goulatis
- Rasika Ramanathan
Organizations
- Defense Threat Reduction Agency
- University of Wisconsin–Madison