Recombinant Escherichia coli Triosephosphate isomerase (tpiA)
Description
Definition and Role in Glycolysis
Triosephosphate isomerase (TPI) catalyzes the reversible conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (GAP) in glycolysis and gluconeogenesis. In E. coli, the gene tpiA encodes this enzyme, which is essential for glucose metabolism . Recombinant TPI retains its native enzymatic activity when expressed in E. coli, making it a valuable tool for studying glycolytic pathways and enzyme engineering .
Expression and Purification
Recombinant E. coli TPI is typically expressed using T5 or T7 promoters in vectors like pQE30 . High-yield expression (>95% purity) is achieved through:
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Induction with IPTG (0.1–1 mM).
Table 2: Recombinant TPI Expression Parameters
| Host Strain | Media | Induction OD₆₀₀ | Yield (mg/L) |
|---|---|---|---|
| M15 | LB | 0.6 | 12.5 |
| DH5α | M9 | 0.1 | 8.7 |
Biochemical Properties
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Thermostability: Recombinant TPI retains activity at 90°C, with a half-life of 280 min (e.g., Pyrococcus woesei TpiA) .
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Metabolic Impact: Overexpression in E. coli alters host metabolism, upregulating chaperones (e.g., DnaK) and glycolytic enzymes .
Table 3: Protein Interactions in E. coli Glycolysis
Applications in Research
Recombinant TPI is used to:
Challenges and Considerations
Product Specs
Target Background
KEGG: ecd:ECDH10B_4108
