One-Step Purification and N-Terminal Functionalization of Bioactive Proteins Via Atypically Split Inteins

Overview

Journal Bioconjug Chem

Publisher American Chemical Society

Specialty Biochemistry

Date 2024 May 30

PMID 38815180

Authors

Ryan Gharios

Annabella Li

Irina Kopyeva

Ryan M Francis

Cole A DeForest

Affiliations

Soon will be listed here.

Abstract

Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our method─referred to as VidaL-tagged expression and protein ligation (VEPL)─enables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (β-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.

References

Keeble A, Turkki P, Stokes S, Khairil Anuar I, Rahikainen R, Hytonen V . Approaching infinite affinity through engineering of peptide-protein interaction. Proc Natl Acad Sci U S A. 2019; 116(52):26523-26533. PMC: 6936558. DOI: 10.1073/pnas.1909653116. View

Reyes-Turcu F, Horton J, Mullally J, Heroux A, Cheng X, Wilkinson K . The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell. 2006; 124(6):1197-208. DOI: 10.1016/j.cell.2006.02.038. View

Ludwig C, Schwarzer D, Zettler J, Garbe D, Janning P, Czeslik C . Semisynthesis of proteins using split inteins. Methods Enzymol. 2009; 462:77-96. DOI: 10.1016/S0076-6879(09)62004-8. View

Weeks A, Wells J . N-Terminal Modification of Proteins with Subtiligase Specificity Variants. Curr Protoc Chem Biol. 2020; 12(1):e79. DOI: 10.1002/cpch.79. View

Keeble A, Yadav V, Ferla M, Bauer C, Chuntharpursat-Bon E, Huang J . DogCatcher allows loop-friendly protein-protein ligation. Cell Chem Biol. 2021; 29(2):339-350.e10. PMC: 8878318. DOI: 10.1016/j.chembiol.2021.07.005. View

Gilmore J, Scheck R, Esser-Kahn A, Joshi N, Francis M . N-terminal protein modification through a biomimetic transamination reaction. Angew Chem Int Ed Engl. 2006; 45(32):5307-11. DOI: 10.1002/anie.200600368. View

Mootz H . Split inteins as versatile tools for protein semisynthesis. Chembiochem. 2009; 10(16):2579-89. DOI: 10.1002/cbic.200900370. View

Obermeyer A, Jarman J, Francis M . N-terminal modification of proteins with o-aminophenols. J Am Chem Soc. 2014; 136(27):9572-9. PMC: 4353012. DOI: 10.1021/ja500728c. View

Shadish J, Benuska G, DeForest C . Bioactive site-specifically modified proteins for 4D patterning of gel biomaterials. Nat Mater. 2019; 18(9):1005-1014. PMC: 6706293. DOI: 10.1038/s41563-019-0367-7. View

10.

Rosen C, Francis M . Targeting the N terminus for site-selective protein modification. Nat Chem Biol. 2017; 13(7):697-705. DOI: 10.1038/nchembio.2416. View

11.

Burton A, Haugbro M, Parisi E, Muir T . Live-cell protein engineering with an ultra-short split intein. Proc Natl Acad Sci U S A. 2020; 117(22):12041-12049. PMC: 7275667. DOI: 10.1073/pnas.2003613117. View

12.

Fisher S, Baker A, Shoichet M . Designing Peptide and Protein Modified Hydrogels: Selecting the Optimal Conjugation Strategy. J Am Chem Soc. 2017; 139(22):7416-7427. DOI: 10.1021/jacs.7b00513. View

13.

Thompson R, Muir T . Chemoenzymatic Semisynthesis of Proteins. Chem Rev. 2019; 120(6):3051-3126. PMC: 7101271. DOI: 10.1021/acs.chemrev.9b00450. View

14.

Prabhala S, Gierach I, Wood D . The Evolution of Intein-Based Affinity Methods as Reflected in 30 years of Patent History. Front Mol Biosci. 2022; 9:857566. PMC: 9033041. DOI: 10.3389/fmolb.2022.857566. View

15.

Friedel K, Popp M, Matern J, Gazdag E, Thiel I, Volkmann G . A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine. Chem Sci. 2019; 10(1):239-251. PMC: 6333167. DOI: 10.1039/c8sc01074a. View

16.

Liebscher S, Schopfel M, Aumuller T, Sharkhuukhen A, Pech A, Hoss E . N-terminal protein modification by substrate-activated reverse proteolysis. Angew Chem Int Ed Engl. 2014; 53(11):3024-8. DOI: 10.1002/anie.201307736. View

17.

Taylor R, Geeson M, Journeaux T, Bernardes G . Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation. J Am Chem Soc. 2022; 144(32):14404-14419. PMC: 9389620. DOI: 10.1021/jacs.2c00129. View

18.

Gharios R, Francis R, DeForest C . Chemical and Biological Engineering Strategies to Make and Modify Next-Generation Hydrogel Biomaterials. Matter. 2024; 6(12):4195-4244. PMC: 10836217. DOI: 10.1016/j.matt.2023.10.012. View

19.

Thiel I, Volkmann G, Pietrokovski S, Mootz H . An atypical naturally split intein engineered for highly efficient protein labeling. Angew Chem Int Ed Engl. 2014; 53(5):1306-10. DOI: 10.1002/anie.201307969. View

20.

Ceballos-Alcantarilla E, Merkx M . Understanding and applications of Ser/Gly linkers in protein engineering. Methods Enzymol. 2021; 647:1-22. DOI: 10.1016/bs.mie.2020.12.001. View