Part:BBa_K5189000
metF
metF
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Origin E. coli; synthetic
Properties
Properties
The metF gene encodes the 5,10-methylenetetrahydrofolate reductase (MTHFR), an enzyme crucial in the folate metabolic pathway.
Usage and Biology
The metF gene encodes the 5,10-methylenetetrahydrofolate reductase MTHFR, which plays a vital role in the folate metabolic pathway. By overexpressing the intrinsic enzymes dihydrofolate reductase (DHFR) and methylene-THF dehydrogenase (MTHFR), and introducing the genes encoding enzymes formate-THF ligase (FTHFL), formyl-THF cyclohydrolase (FTHFC), and MTHFD from the one-carbon metabolic pathway of Methylobacterium extorquens AM1 or Clostridium autoethanogenum, an additional pathway was constructed upon the native pathway to produce L-5-MTHF.
Cultivation, Purification, and SDS-PAGE
The metF gene was successfully amplified using PCR, yielding bands of 894bp, as expected. The gene was inserted into the pRSFduet-1 vector using BamHI and HindIII digestion. The recombinant plasmid was transformed into E. coli DH5α, and validation was performed through colony PCR and enzyme digestion. The expected band sizes confirmed successful ligation, as shown in the gel electrophoresis results.
The pRSFDuet-metF-folA plasmid was transformed into E. coli BL21(DE3) to evaluate the co-expression of the metF and folA genes. Protein expression was induced using IPTG and analyzed via SDS-PAGE and Western Blot techniques. The SDS-PAGE results displayed distinct bands corresponding to the metF protein, particularly under induction at 37°C. Western Blot analysis confirmed the successful expression of all proteins, demonstrating effective co-expression.
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