Cloning of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase Gene from Vanda Mimi Palmer and Its Heterologous Expression in Escherichia coli
Bibliographic record
Abstract
Plant 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is involved in the conversion of HMG-CoA into mevalonate (MVA), which yields a biologically active isoprenoid precursor, isopentenyl pyrophosphate (IPP) unit. To date, heterologous expression of HMGR isolated from orchidaceae family has not been reported. The aims of this study were to isolate, clone, over-express and functionally characterize the cDNA encoding a 3-hydroxy-3-methylglutaryl-CoA-reductase of Vanda Mimi Palmer (VMPHMGR) in Escherichia coli strain BL21 (DE3) pLysS. The deduced VMPHMGR contains an open reading frame (ORF) of 1689 bp and generates a protein of 562 amino acids with a calculated molecular mass of 59780 Da and a predicted pI value of 6.63, which is 76% identical to other plant HMGRs. Expression analysis of VMPHMGR transcript by real-time RT-PCR showed that it was differentially regulated. Primers with appropriate restriction sites were used to amplify and facilitate in-frame cloning of the VMPHMGR into pET32(a). The expression of VMPHMGR, in E. coli, fused to N-terminal thioredoxin (Trx·Tag), S·Tag and His·Tag fusion proteins in pET32(a) yielded a partially soluble recombinant protein. This expressed VMPHMGR was subjected to functional enzymatic assay and GCMS analysis of the end products detected dehydromevalonic lactone and pantolactone, which were derivatives of mevalonate lactone.
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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".