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Updated: Aug 3, 2026
07:11
Homemade Site Directed Mutagenesis of Whole Plasmids
Published on: May 11, 2009
Site-directed mutagenesis with an expanded genetic code
D Mendel1, V W Cornish, P G Schultz
1Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285-0540, USA.
Annual Review of Biophysics and Biomolecular Structure
|January 1, 1995
Summary
This study introduces a biosynthetic method for site-specifically incorporating diverse amino acids into proteins. This technique allows precise protein modifications to investigate structure-function relationships and create labeled proteins for biophysical studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Site-specific incorporation of amino acids into proteins is crucial for understanding protein structure-function relationships.
- Existing methods have limitations in the diversity and specificity of amino acid incorporation.
Purpose of the Study:
- To develop a versatile biosynthetic method for site-specific incorporation of a wide range of amino acids and analogues into proteins.
- To utilize this method for probing protein stability, enzyme mechanisms, and creating specifically labeled proteins for biophysical analysis.
Main Methods:
- Utilizing an amber suppressor transfer RNA (tRNA) aminoacylated with a desired amino acid.
- Introducing an amber codon into the protein's DNA sequence for site-specific incorporation.
- Employing various amino acid analogues including fluorinated tyrosines, hydrophobic amino acids, glutamate analogues, and conformationally restricted amino acids.
Main Results:
- Demonstrated successful site-specific incorporation of numerous amino acid analogues into proteins.
- Investigated the impact of hydrogen bonding and hydrophobic packing on protein stability using specific amino acid substitutions.
- Probed enzyme mechanisms (staphylococcal nuclease, ras) and constructed proteins with photoaffinity, spin, and isotopic labels.
Conclusions:
- The developed biosynthetic method offers a powerful tool for precise protein engineering and functional studies.
- This technique enables detailed structure-function investigations and the creation of custom-labeled proteins for advanced biophysical research.
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