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Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...

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High-throughput T7 LIC vector for introducing C-terminal poly-histidine tags with variable lengths without extra

Jonas Lee1, Sung-Hou Kim

  • 1Department of Chemistry, University of California, Berkeley, California 94720-5230, USA.

Protein Expression and Purification
|October 1, 2008
PubMed
Summary

Researchers modified an expression vector for easier cloning of proteins with varying histidine tag lengths, improving purification via immobilized metal ion affinity chromatography (IMAC). This facilitates testing multiple His-tag lengths for challenging protein targets.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Immobilized metal ion affinity chromatography (IMAC) is a key method for purifying recombinant proteins using histidine tags (His-tags).
  • Some proteins exhibit weak binding to IMAC resins, leading to low purity, often necessitating longer His-tags (8-10 histidines).
  • Existing expression vectors offer limited flexibility for easily cloning and testing diverse His-tag lengths.

Purpose of the Study:

  • To engineer a versatile expression vector for streamlined cloning and testing of various His-tag lengths.
  • To address the limitations of current vectors in facilitating His-tag length optimization for challenging proteins.
  • To enable efficient parallel cloning of target genes with different His-tag constructs.

Main Methods:

  • Modification of the Escherichia coli T7 expression vector pET21a.
  • Incorporation of ligation-independent cloning (LIC) into the vector.
  • Engineering of N-terminal and C-terminal linkers to ensure precise His-tag incorporation without extraneous sequences.

Main Results:

  • Developed a modified pET21a vector enabling easy and efficient parallel cloning of genes with varying His-tag lengths.
  • The engineered LIC system allows for the use of a single insert to generate multiple His-tag constructs.
  • The vector design prevents the translation of unwanted amino acid sequences, ensuring clean His-tag fusion.

Conclusions:

  • The novel LIC-enabled vector provides a flexible and efficient tool for optimizing His-tag length in protein purification.
  • This approach simplifies the process of improving the purity of difficult-to-purify recombinant proteins using IMAC.
  • Facilitates rapid screening of His-tag lengths for enhanced protein binding and purity in IMAC.