Recombinant Pongo pygmaeus Hemoglobin subunit delta (HBD)
Description
Production and Engineering
The protein is produced in yeast, a cost-effective eukaryotic system enabling post-translational modifications like glycosylation while maintaining high yield . Key steps include:
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Gene Cloning: The HBD gene fragment (AA 2–147) is fused with an N-terminal 6xHis-GST tag for purification .
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Expression: Optimized yeast fermentation ensures proper folding and solubility.
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Purification: Affinity chromatography using the His tag achieves >90% purity .
Transcriptional Regulation
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Editing the HBD promoter with KLF1, β-DRF, and TFIIB motifs increased HBD expression to 46% of total β-like globins in erythroblasts, demonstrating therapeutic potential for β-hemoglobinopathies .
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DNA-PKcs inhibitors (e.g., AZD-7648) enhanced homology-directed repair (HDR) efficiency, boosting HBD levels in hematopoietic stem/progenitor cells (HSPCs) .
Functional Insights
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In murine models, activated δ-globin expression improved β-thalassemia phenotypes, validating HBD as a therapeutic target .
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HPLC confirmed that increased HBD transcription correlates with elevated HbA2 protein levels (from <5% to 30% in edited clones) .
