Abstract
Although the use of affinity tags can greatly improve purification of expressed enzymes, the placement of affinity tags can significantly impact the expression, solubility, and function of recombinant proteins. To facilitate the optimal design of 6xHis-tagged constructs for protein purification, we developed Terminator, a Python-based software package, which takes a UniProt ID or existing protein sequence as input, identifies related sequences, maps sequence conservation retrieved from ConSurf onto protein 3D structures retrieved from the PDB and SWISS-MODEL, and analyzes proximity to cavities and functional sites to recommend the N- or C-terminus for placement of 6xHis fusion tags 86-100% accuracy in predicting the relative risk of ill effects between termini and a 92-93% accuracy in predicting the absolute risk of modifying individual termini. This reliability of Terminator's analysis suggests that proximity to surface cavities, not burial of wild-type termini, is the most reliable predictor of ill effects arising from short 6xHis fusion tags. This tool aims to expedite construct design and enhance the successful production of well-behaved proteins for studies in enzymology and biocatalysis with minimal need for computational resources, programming knowledge, or familiarity with protein-tag interference mechanisms.
Recommended Citation
R. Gerulskis and S. D. Minteer, "Terminator: A Software Package for Fast and Local Optimization of His-Tag Placement for Protein Affinity Purification," ACS Bio and Med Chem Au, vol. 5, no. 1, pp. 55 - 65, American Chemical Society, Feb 2025.
The definitive version is available at https://doi.org/10.1021/acsbiomedchemau.4c00055
Department(s)
Chemistry
Publication Status
Open Access
Keywords and Phrases
6xHis tag; affinity purification; biocatalysis; bioinformatics tool; ConSurf; enzymology; protein design
International Standard Serial Number (ISSN)
2694-2437
Document Type
Article - Journal
Document Version
Final Version
File Type
text
Language(s)
English
Rights
© 2025 American Chemical Society, All rights reserved.
Creative Commons Licensing
This work is licensed under a Creative Commons Attribution 4.0 License.
Publication Date
19 Feb 2025
Comments
Office of Naval Research, Grant N000142114008