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Related Experiment Video

Updated: Jan 17, 2026

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Published on: January 26, 2017

Optimal TELSAM-Target Protein Linker Character is Target Protein-Dependent.

Maria Jose Pedroza Romo¹, Alihikaua Keliiliki¹, Jacob C Averett¹

¹Brigham Young University-Provo: Brigham Young University.

Biorxiv : the Preprint Server for Biology

|September 15, 2025

Summary

This summary is machine-generated.

Optimizing protein crystallization involves selecting the right linker between the TELSAM chaperone and target proteins. Linker choice and the presence of a His tag significantly impact crystal quality and diffraction limits for structural studies.

Keywords:

DARPin Designed Ankyrin Repeat Protein ETV6 TELSAM linkers protein binding

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Application of I TASSER, trRosetta, UCSF Chimera, HADDOCK server, and HEX loria for De Novo and In Silico Design of Proteins

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Published on: July 8, 2025

Area of Science:

Structural Biology
Biochemistry
Crystallography

Background:

The sterile alpha motif domain (TELSAM) of the human translocation ETS leukaemia protein enhances protein crystallization by forming stable lattices.
Crystal quality and diffraction limits depend heavily on linker choice and the N-terminal 10xHis tag on TELSAM.
Current methods for identifying optimal linkers are often trial-and-error.

Purpose of the Study:

To systematically evaluate different linker types and lengths for fusing TELSAM to target proteins.
To assess the impact of the N-terminal 10xHis tag on TELSAM-mediated protein crystallization.
To identify optimal linker strategies for improved protein crystallization and high-resolution structure determination.

Main Methods:

Designed and constructed multiple fusion proteins with varying linkers (rigid, semi-flexible, flexible) between TELSAM and target proteins (DARPin, TNK1 UBA domain).
Constructs were made with and without the N-terminal 10xHis tag.
Crystallization propensity, crystal size, morphology, and diffraction quality were analyzed.

Main Results:

Short semi-flexible and rigid linkers yielded large crystals rapidly with a DARPin target.
Flexible linkers were optimal for the TNK1 UBA domain target protein.
Removing the 10xHis tag generally improved crystallization rates, morphology, and propensity.
His tag removal enhanced diffraction limits and crystal quality for specific constructs.

Conclusions:

Linker selection is crucial and protein-dependent for successful TELSAM-mediated crystallization.
Short, flexible, or semi-flexible linkers are recommended for optimal protein crystallization.
Removing the His tag can improve crystallization outcomes, aiding high-resolution structure determination.