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Updated: Jun 15, 2026

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
10:36

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Published on: July 27, 2016

Cleavable C-terminal His-tag vectors for structure determination.

William H Eschenfeldt1, Natalia Maltseva, Lucy Stols

  • 1Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Bldg. 202/Rm. BE111, 9700 South Cass Avenue, Argonne, IL 60439, USA.

Journal of Structural and Functional Genomics
|March 10, 2010
PubMed
Summary

Researchers developed new expression vectors to determine protein structures. These vectors use cleavable C-terminal tags, successfully determining structures for proteins that previously failed standard N-terminal tagging methods.

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Area of Science:

  • Structural biology
  • Genomics
  • Protein science

Background:

  • High-throughput structural genomics aims to map protein structure space.
  • Standard protocols often use N-terminal His-tags for protein purification.
  • Many proteins fail standard protocols but warrant further investigation.

Purpose of the Study:

  • To develop alternative strategies for determining protein structures.
  • To salvage proteins that fail conventional N-terminal tagging methods.
  • To improve success rates in structural genomics projects.

Main Methods:

  • Development of ligation-independent cloning expression vectors with cleavable C-terminal tags.
  • Utilizing C-terminal His(6)-tag and His(6)- and MBP-tags.
  • Employing TEV or both TEV and TVMV proteases for tag cleavage.

Main Results:

  • Successful structure determination for 5 out of 16 proteins that failed N-terminal tagging.
  • Demonstrated efficacy of C-terminal tagging as a salvage pathway.
  • One protein structure was determined after multiple alternative salvaging methods failed.

Conclusions:

  • Novel C-terminal tagging vectors offer a viable alternative for structural genomics.
  • These vectors enhance the recovery of valuable protein structures.
  • The approach expands the scope of proteins amenable to structural determination.