Part:BBa_K2856001
Bifunctional glutamate-cysteine ligase/glutathione synthase (gshF)
The BBa_K2856001 harbors a coding sequence of bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) derived from S.agalactiae. Codon-optimization has been made for Lactococcus Lactis. gshFp catalyzes the conversion of Cys,Glu and Gly to GSH.
Usage and Biology
Bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) is an enzyme involved and responded to synthetic reaction of GSH.In this reaction,one Cys and one Glu are converted to one γ-GC,then one γ-GC and one Gly are converted to one GSH.(Figure 1) The Lactococcus Lactis NZ9000 has inability to synthesis GSH.In our project,we construct a plasmid harboring gshF in order to produnce GSH in Lactococcus Lactis NZ9000.
Protein Analysis
SDS-PAGE was performed to detect the protein expression level of gshF. The cells were washed twice with 0.1 M PBS after centrifugation.Crude protein was extracted through cell breaking using ultrasonication and centrifugation. Then the samples were added into gel to analysis the protein.
Validation of GSH by HPLC analysis
To further confirm the synthetic GSH in Lactococcus lactis, HPLC was performed to analyze the product. GSH was identified on the basis of retention times related to standard sample. According to the retention time of standard GSH sample, GSH compound of extracts from engineered Lactococcus lactis can be confirmed.Compared with engineered strain,we didn't detect GSH in wild type Lactococcus lactis NZ9000.
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]
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