Design

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.

The sequence for the design is retrieved 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 pET-28a(+) 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. With Colony PCR and Gradient PCR the successful incorporation of the G-block into the vector is verified.

In this engineering cycle we prepare the plasmid used for expression, transform cells to an expression strain, and verify the successful cloning to continue expression and purification of GR.

Build

The GR must be prepared for expression in a plasmid and with the His-tag right after the thrombin cleavage site (Simmons et al. 2008). The sequence can be found in the RCSB Protein Data Bank and the protocols are from He et al. 2016.

The sequence was then prepared by identifying the required LBD with the Bledsoe et al. 2002 publication, introducing mutations that are improving solubility; F602A, C622Y, T668V, S674T, V675I (He et al. 2014) and adding the His-tag based on Simmons et al. 2008 and He et al. 2014 protocols. From the European Nucleotide Archive by EMBL-EBI (entry X03225.1) the mRNA sequence for the protein of interest was retrieved. After rare codon analysis with GenScript Rare Codon Analysis Tool and codon optimisation for expression in E. coli with Sequence Optimizer by Twist Bioscience of the region of interest, flanking sites for introduction into the pET-28a(+) vector were added. The complete sequence is called the G-block.

In the wet lab experiments the pET-28a(+) vector and G-Block were inserted into the Top10 competent E.coli culture. Later on the plasmids were transferred to a protein expression strain of BL21(DE3) Competent E. coli. The components for the cloning procedure were the G-Block plasmid prepared by us and produced by Integrated DNA Technologies, and reagents provided by the facilities of the EvoGene research division at the University of Oslo.

The bacteria was cultured with kanamycin as a selection marker for successful transformation as the pET-28a(+) plasmid contains a kanamycin resistance gene. Additionally, Colony PCR and Gradient PCR were performed to verify correct cloning resulting in successful plasmid preparation.

Test

For a successful engineering cycle, the linearisation of constructs, vector, and proper integration of the G-block into the pET-28a(+) vector must be verified, as well as proper transformation of Top10 competent E.coli.

A first indicator for correct assembly of the vector and G-Block was the survival of cells in kanamycin culture. However, to exclude the possibility of random mutations towards antibiotic resistance other verification experiments were conducted. Using Colony PCR, Gradient PCR, enzyme digestion, and agarose gel analysis, the correct band size for the plasmid was visible. The results are captured as pictures. (see Results)

Learn

The learning outcome aimed for in this cycle was the preparation of plasmids and all required components for said plasmid construct. Due to the essential information from He et al. 2014 about sequence optimisation, the plasmid preparation went successful and the experimental procedure could be continued with expression and purification of the GR. The results are analyzed by the team with guidance from the team’s supervisors.

Design

In this engineering cycle, the GR is expressed and purified. The resulting protein may be found in the soluble, intermediate, or solid fraction and will be investigated by using all of them separately. The expression strain of this engineering cycle is the BL21(DE3) Competent E. coli cells that have been transformed with the pET28-His-GR plasmid construct prepared with pET-28a(+) as the vector, and the G-Block carrying the GR LBD sequence with His-tag and flanking sites for insertion into the vector. The protein isolation samples are tested for different conditions to find the best suitable fraction for GR purification.

Build

The BL21(DE3) Competent E. coli cells were cultured in kanamycin-containing broth to remove any bacteria without the resistance gene that is provided by the pET28-His-GR plasmid construct. During the first engineering cycle, the existence of the correct plasmid was verified with PCRs and agarose gel electrophoresis. Following the cultivation, the cell samples were prepared with DNAse and lysed to gather the protein. With careful centrifugation with nickel beads to bind the protein, first a soluble phase from the supernatant and a solid protein fraction from the pellet were collected. Lastly, the remaining supernatant was processed with syringe-passing for filtration. The three phases were next tested with SDS PAGE to detect GR.

The components for this lab procedure revolving GR expression were the DNA-plasmid previously prepared by us (see 1st engineering cycle) and reagents available for research use from the facilities of the EvoGene research division at the University of Oslo.

Test

It was essential to determine the purification step that yields GR most efficiently. This was done by comparing the protein samples gathered from the supernatant (soluble protein), the pellet (solid phase protein/insoluble), and the syringe-filtered phase by SDS PAGE. The outcome of this experimental procedure did not show protein yields as expected, as they were generally too low for further experimentation (see Results). These results urged for troubleshooting.

To improve the protein purification outcome, a different method was used. With a chromatographic separation procedure, three phases were obtained: a flowthrough phase, a washing phase, and an elution phase. In chromatographic experiments, the components in the sample are separated due to their different affinities to the stationary phase. Flowthrough samples have a very low binding affinity, while the elution phase samples have a high affinity.

Following the purification by chromatography, a concentration procedure of the samples was done to increase the amount of protein per volume, as it was too low for SDS PAGE previously.

GR is expected to be observed in the elution phase. SDS PAGE followed by Western Blotting was conducted as verification for the experiment. As expected, the protein yield increased, with the GR being found in the elution phase (see Results).

To verify the results as correct, the different phases containing protein are compared with positive and negative controls. For further experimentation on the overall project, a proper quantity of the elution phase containing GR is essential.

Learn

From this second engineering cycle, the adjustment of protocols towards a better protein yield and finding the correct phase for the protein of interest was the learning outcome. In the test phase, it was shown that nickel bead purification was unsuited for GR. However, chromatographic separation followed by a procedure to increase concentration resulted in evidence of GR in the expected elution phase sample on the Western Blot. This realisation is crucial for further experimentation and optimisation. However, since the protein concentration needed to be increased before analysis, optimisation of protein expression targeting the induction in the E. coli would be a natural further improvement of the experimental protocols.