Expression and purification of active protein kinase Cd (PKCd) kinase domain from E. coli
Bibliographic record
Abstract
PKC isozymes can be divided into three subfamilies based on structural and enzymatic differences: conventional, novel and atypical. PKCδ is a member of the subfamily of novel PKC's. In an effort to obtain X‐ray crystal structure of this nPKC family target, we expressed and purified a recombinant PKCδ kinase domain construct in E. coli. Here we show that the recombinant kinase domain has activity, is monomeric by size‐exclusion chromatography (SEC), and can auto‐phosphorylate heterogeneously within the cell during expression. PKCδ kinase domain (317–676) was expressed in BL21 cells with a C‐terminal 6‐His tag, and purified +/− ATP to >95% following a four‐step protocol. The purified product eluted as a single, monomeric peak during SEC. Multiple phosphorylations (avg 5–7) were observed by ESI‐MS for both of the +/− ATP preparations; indicating that all phosporylation events most likely occur during expression. In addition, both +/− ATP preparations displayed substrate inhibition with the PKC‐specific FARK inhibitor peptide, while NMR saturation transfer difference (STD) binding experiments showed that AMP‐PNP binds and competes with bisindolylmaleimide for the same active site of PKCδ (317–676). Although, the construct proved difficult to crystallize, we have successfully produced a method for purification of a PKCδ kinase domain from E. coli suitable for assay development, and selectivity studies with other nPKC family members.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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".