Part:BBa_K4171002

SelA

Background

SelA is an enzyme used in natural selenocysteine (Sec) synthesis pathway. Its function is to convert seryl-tRNA into selenocysteinyl-tRNA with selenophosphate provided by SelD (BBa_K4171003).

Fig. 1. In vitro Sec synthesis pathway

Usage

SelA (BBa_K4171002) was overexpressed in E. coli MG1655 and purified by His-Tag protein purification column. Then, purified SelA was used to convert seryl-tRNA into selenocysteinyl-tRNA.

Characterization

SDS-PAGE analysis was conducted to confirm the success of SelA (BBa_K4171002) purification.

Fig. 2. Confirmation of SelA (BBa_K4171002) and SelD 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 SelA (BBa_K4171002) 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 tRNA^Ser (BBa_K4171007); (B) Sec synthesized by tRNA^UTuX (BBa_K4171009).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

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• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]