Part:BBa_K4291006

Added by BFSU-ICUnited

Analysis of tyrosinase expression and surface localization

To confirm the expression of tyrosinase on the cell-surface of E. coli, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed according to a previous study. The strains were grown in Luria-Bertani (LB) medium at 37 ◦C for 16–20 h and then centrifuged at 8000 rpm for 10 min. The harvested cells were suspended in Tris-HCl buffer (20 mM Tris, pH 8.0). Whole cells (T) were prepared by sonication for 30 min at 4 ◦C in 10 mM Tris-HCl pH 8.0 buffer and then centrifuged at 8000 rpm. The supernatant served as the cytoplasmic fraction (P). The sediment was further treated with N-lauryl sarcosyl at a final concentration of 2% at room temperature and then centrifuged at 16,000 rpm for 60 min to separate the inner and outer membranes (Beckman, Germany). All samples were run on 15% (w/v) SDS-PAGE gels. Western blot was performed using His-tag and goat anti-mouse IgG as the primary and secondary antibodies,respectively.

Figure 1

Expression of the InaK-Tyr fusion protein in E. coli-Tyr was assessed by subcellular fractionation followed by western blotting and SDS-PAGE. In the outer membrane fraction, a band matching the InaK-Tyr fusion protein (approximately 84 kDa) was observed 。

inaK

inaK

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 104
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 294