Part:BBa_K5333000

pET28-His-GR

pET28-His-GR is a coding sequence that is used for expressing the ligand binding domain of the glucocorticoid receptor using Escherichia coli vector pET-28a(+).

This coding sequence will allow to express the LBD of Glucocorticoid Receptor from the vector including a his-tag for purification and attachment to an electrode for later ligand-binding measurements.

Usage and Biology

The purpose of our design is to express the glucocorticoid receptor (GR) with a His-tag for purification from Escherichia coli cells. The GR must be prepared for expression from the vector including a his-tag for purification and attachment to an electrode for later ligand-binding measurements. The prepared sequence is then used to transform the expression strain. For the design the sequence is used from online databases such as UniProt and the RCSB Protein Data Bank. The protocols by He et al. 2014 are used as guidance for the build. In the wet lab for cloning the construct Top10 E.coli competent cells are used. The G-block sequence pET-28-His-GR is ordered from Integrated DNA Technologies (Ref# 239413871) and cloned by Gibson Assembly into the linearised pET28(+) vector, which is from Novagen, carries as kanamycin resistance gene, requires as T7 polymerase for expression, and has a thrombin cleavage site right after an n-terminal His-tag.

In order to fit the requirements for the addition of New Basic Parts excluding the potential illegal parts in our sequence, we added silent mutations on the restriction enzyme cleavage sites on the sites 103, 304 and 559. While the reverse complement sequence translated to amino acids remains the same, the forward sequence translation has a single change in Q99E which means that in the position 99 a Q (Glutamin) changes to E (Glutamic acid). A second aminoacid also changes in the forwards sequence translation in F252v, which meand that in the position 252 a F (Phenylalanine) changes to V (Valine)

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