Part:BBa_K4785005:Design

high-mobility group box 1 (hmgb1)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 73
  

Design Notes

In order to examine the functionality of the HMGB1 protein and its domains, the subsequent four plasmids were designed to carry out protein expression.

Figure 1: To purify the target proteins, we construct five plasmids: pET28a-6×His-HMGB1_FL, pET28a-6×His-HMGB1_AB, pET28a-6×His-HMGB1_A, pET28a-6×His-HMGB1_B. After sequencing, we determine that the plasmid was correctly sequenced and ready for use.

Figure 2: The plasmids with the correct sequencing results were transferred into Escherichia coli BL21 for induced expression, then Escherichia coli was broken, and the target protein was purified by affinity chromatography and molecular sieve, and we obtained high purity of the target protein. (a), SDS-PAGE of HMGB1_A. (b), SDS-PAGE of HMGB1_B. (c), SDS-PAGE of HMGB1_AB. (d), SDS-PAGE of HMGB1_FL.

HMGB1_A and HMGB1_B appear to be more highly expressed in E. coli, presumably due to their shortened sequence lengths and reduced number of modifications, as determined experimentally.

Source

Organism: Homo sapiens