Protein purification methods employing tandem affinity purification (TAP) tags have become an increasingly useful tool to gain information about the composition of cellular protein complexes and interactions among proteins. Generally, TAP tag constructs consist of two tandemly repeated Staphylococcus aureus protein A domains, one TEV protease cleavage site and a small peptide comprising a calmoduline binding domain. Originally designed for yeast expression, when expressed in the filamentous fungus Aspergillus nidulans detectable levels of TAP tag fusion proteins are rather low owing to the species' codon usage. By application of site directed mutagenesis, we have altered all the rarely used codons in the commonly employed TAP tag construct to achieve higher rates of translation in the endogenous host. Both versions of the tag suited for N-terminal and C-terminal fusions were modified. Expression levels of these modified TAP tags were tested by construction of fusions to the green flourescent protein (GFP), the expression of which was driven by the inducible alcA promoter. After functionality could be validated, chimeric constructs with one regulator of A. nidulans fruit body formation were expressed and a purification protocol for complex enrichment could be established.
Full conference title:
23rd Fungal Genetics Conference
- Fungal Genetics Conference 23rd (2002)