Part:BBa_K4171003
SelD
Background
SelD is an selenophosphate synthetase used in the natural selenocysteine (Sec) synthesis pathway. It functions to synthesize selenophosphate from selenide and ATP, offering the sources of selenium which can be used by SelA (BBa_K4171002) to form selenocystinyl-tRNA.
Fig. 1. In vitro Sec synthesis pathway
Usage
SelD (BBa K4171003) was overexpressed in E. coli MG1655 and purified by His-Tag protein purification column. Then, purified SelD (BBa K4171003) was used to synthesize selenophosphate from sodium selenite.
Characterization
SDS-PAGE analysis was conducted to confirm the success of SelD (BBa K4171003) purification.
Fig. 2. Confirmation of SelA and SelD (BBa K4171003) purification by SDS-PAGE. M: Marker; Lane 1: SelD before condensing (~36kDa); Lane2: SelA before condensing (~50kDa); Lane3: SelD after condensing; Lane4: SelA after condensing.
To verify SelD (BBa K4171003) function, HPLC was performed to examine whether Sec is produced. In the HPLC results, the retention time of the Sec standard is 10.5 minutes (the peaks at 3.5 minutes and 4.5 minutes are solvent peaks), which is the same as that of Sec synthesized via the in vitro pathway. This proves that the enzyme functions ideally.
Fig. 3. Confirmation of Sec by HPLC. (A) Sec synthesized by tRNASer (BBa_K4171007); (B) Sec synthesized by tRNAUTuX (BBa_K4171009).
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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