Single-step purification of intrinsic protein complexes in Saccharomyces cerevisiae using regenerable calmodulin resin
Bibliographic record
Abstract
Characterization of protein- protein interactions is a vital aspect of molecular biology as multi-protein complexes are the functional units of cellular processes and defining their interactions provides valuable insights into their function based on a “guilt by association” concept. To identify the components in complexes, purification of the latter near homogeneity is required. The tandem affinity purification (i.e., TAP) method, coupled with mass spectrometry have been extensively used to define native protein complexes and transient protein-protein interactions under near physiological conditions (i.e., conditions approximate of the internal milieu) in Saccharomyces cerevisiae. Generally, TAP consists of two-stage protein enrichment using dual affinity tags, a calmodulin-binding peptide and a Staphylococcus aureus protein-A, separated by a tobacco etch virus protease site, which are fused to either the C- or N-terminal of the target protein. TAP-tagging has proved to be a powerful method for studying functional relationship between proteins and generating large-scale protein networks. The method described in this paper provides an inexpensive single-step purification alternative to the traditional two step affinity purification of TAP-tagged proteins using only the calmodulin-binding peptide affinity tag. Moreover, a novel protocol for the regeneration of the calmodulin-agarose resin is outlined and validated. This basic approach allows fast and cost-effective purification of proteins and their interacting partners from Saccharomyces cerevisiae.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".