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Related Experiment Videos

UGA read-through artifacts--when popular gene expression systems need a pATCH

G MacBeath1, P Kast

  • 1Scripps Research Institute, La Jolla, CA, USA.

Biotechniques
|May 20, 1998
PubMed
Summary
This summary is machine-generated.

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The TGA stop signal in pET vectors causes unwanted protein extension. A new plasmid, pATCH1, corrects this issue, ensuring pure protein preparations for researchers.

Area of Science:

  • Molecular Biology
  • Protein Expression
  • Genetics

Context:

  • pET vectors are standard for high-level protein expression in Escherichia coli.
  • C-terminal histidine (His) tags are commonly used for protein purification.
  • The TGA sequence following the His-tag can lead to translational read-through.

Purpose:

  • To investigate the cause of heterogeneous protein preparations from pET constructs.
  • To identify the mechanism of read-through at the TGA stop codon.
  • To develop a solution for achieving homogeneous protein expression.

Summary:

  • Read-through of the TGA stop codon in pET vectors results in a 21-amino acid extension.
  • Mass spectrometry confirms tryptophan insertion at the UGA (opal) codon in non-opal suppressor strains.

Related Experiment Videos

  • Plasmid pATCH1 offers a corrected stop signal sequence to resolve read-through issues.

    • Impact:

    • Highlights the inefficiency of UGA as a termination codon in overexpression vectors.
    • Provides a practical method (pATCH1) to obtain pure protein preparations.
    • Improves the reliability of gene expression and protein purification protocols.