Difference between revisions of "Part:BBa K5333000"

Latest revision as of 21:33, 1 October 2024

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. This variations produced two changes in the translation of the forward sequence, getting a single change in Q99E, a change in the position 99 from Q (Glutamin) changes to E (Glutamic acid); and in the position F252v, a change in F252V, a change in the position 252 a F (Phenylalanine) changes to V (Valine).

We test the expression of our protein (GR-BLD) of interest by doing a standard process of bacterial cloning with the expression vector pET-28a(+) and transformation into E.coli strain BL21(DE3). To show proof of expression we induced the cultures of the transformed strain pET-28-His-GR-BL21(DE3) with the ligand Prednisone and the inducer IPTG, purified the proteins using Ni-NTA Agarose Beads to test if the protein of interest was obtained in the insoluble or soluble fraction of the purification.

We also tested the induction and correct expression of our protein of interest using separation methods of proteins, such as SDS-Page, Chromatography and Western Blot.

This protein can be used in a safety level 2 laboratory.

Sequence and Features