Cell-free synthesis of human 1-adrenergic receptor variants

So far, the majority of in vitro studies with the 1AR have been performed with the turkey receptor, primarily for reasons of instability upon detergent treatment for the human 1AR (Serrano-Vega and Tate 2009). The L-CF-synthesis in presence of NDs avoids any detergent contact and the receptor can be synthesised in a soluble form directly into a relatively defined and homogeneous lipid environment.

The feasibility of the L-CF-synthesis to produce ligand binding competent human 1AR was therefore evaluated.

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The results with the turkey 1AR showed the importance of thermostabilisation for the correct folding of the receptor. Furthermore, both thermostabilised and non-stabilised turkey 1AR have been truncated at the N- and C-terminus as well as in the intracellular loop 3 (ICL 3). Therefore, three variants of the human 1AR were designed. The thermostabilising mutations of the turkey 1AR were transferred to a full-length and a truncated version of human 1AR (ts-fl- 1AR and ts- - 1AR, respectively). Additionally, a truncated receptor variant without potentially thermostabilising mutations was designed ( - 1AR) (see 1.3.4). Like the turkey 1AR, all three variants were designed with a C-terminal sfGFP-tag to simplify the quantification of total receptor yields.

To assure the expression of the DNA-construct in the CECF-system, all three constructs were first synthesised in P-CF mode and analysed by SDS-PAGE and subsequent Coomassie-staining and western-blot and immunostaining of the His-tag (Fig. 4.11).

Fig. 4.11 P-CF production of human 1AR constructs.

Rinsed pellets from P-CF expression of human ts-fl- 1AR-sfGFP-His10, ts- - 1AR-sfGFP-His10 and - 1AR-sfGFP-His10 were resuspended in 100 µl S30 buffer and 1-2 µl were loaded on a 15% SDS-PAGE. Gels were stained with Coomassie (C) or with immunostaining of the His-tag (I) after western blot.

In comparison with the molecular mass marker, ts-fl- 1AR-sfGFP-His10 was found at ca. 85 kDa and both ts- - 1AR-sfGFP-His6 and - 1AR-sfGFP-His6 at ca. 60 kDa in the western-blot. A faint slightly lower protein band was detected in the western-blot for ts-fl- 1AR that might be caused by N-terminal cleavage or unusual running behaviour in PAGE (Rath et al. 2009). The Coomassie-stained SDS-PAGE revealed the co-precipitation of proteins from the RM with the receptors. Distinct receptor bands were found in the Coomassie-stained gel for both deletion constructs, while the according band was relatively faint for ts-fl- 1AR. Expression efficiency might thus be somewhat reduced for the full-length construct.

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Fig. 4.12 Quantity and quality of CF-synthesised human 1AR in ND (DOPG).

Thermostabilised fulllength (tsfl 1AR), thermostabilised truncated (ts 1AR) and nonstabilised truncated ( -1AR) human Adrenergic receptors were CF-synthesised as C-terminal sfGFP-His6 fusion constructs in presence of 60 µM ND (DOPG). After removal of potential precipitates, the total receptor concentrations in the reaction mixtures were determined by the fluorescence of the sfGFP-moieties and the concentration of alprenolol binding sites by radioligand filter binding assay. The fraction of active receptor was calculated assuming one ligand binding site per receptor. Values are given as mean and SD of three independent CF-reactions.

In a next step, soluble yields and ligand binding competence of the constructs were tested after CF-synthesis in presence of 60 µM ND (DOPG) (Fig. 4.12). In accordance to the findings for P-CF CF-synthesis, soluble yield of ts-fl- 1AR was ca. 30% lower than for the truncated thermostabilised receptor variant (11 µM and 16 µM, respectively). The fraction of ligand binding competent receptor was almost similar for both stabilised receptor variants and in a range between 2 and 3%. This corresponds to approximately 270 nM of ligand binding sites (ca. 15 µg/ml) for ts-fl- 1AR-sfGFP-His6 and approximately 510 nM of ligand binding sites (ca. 30 µg/ml) for ts- - 1AR.

L-CF-synthesis of the non-stabilised human 1AR variant appeared to be less efficient than for the thermostabilised constructs, with total soluble yields of only 4.3 µM. Additionally, only 0.8% of this were found to be ligand binding competent, corresponding to ca. 30 nM ligand binding sites in the reaction mixture or roughly 2 µg/ml of ligand binding competent - 1AR-sfGFP-His6.

In a next step, the affinities of the human 1AR constructs towards alprenolol were evaluated.

Therefore, radioligand filter binding assays were performed with the thermostabilised human 1AR variants with constant receptor concentrations and increasing concentrations of ³H-labeled alprenolol (Fig. 4.13). As the concentration of ligand binding sites was insufficiently low for the non-stabilised human 1AR variant, affinity measurements were not performed with this construct.

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Fig. 4.13 Binding affinities of human 1AR constructs in ND (DOPG) to alprenolol.

Truncated (ts- - 1AR) and full-length (ts-fl- 1AR) thermostabilised human 1-adrenergic receptor variants were CF-synthesised as C-terminal sfGFP-His6 fusions in presence of 30 µM ND (DOPG). Dissociation constants (KD) to alprenolol were determined by radioligand filter binding assay using increasing concentrations of ³H-labeled alprenolol and constant receptor concentrations. Values are given as mean and SD of three measurements.

Both truncated and full-length thermostabilised human 1AR show high affinity to alprenolol with KD

of 4.7 nM and 2.0 nM, respectively. This indicates a high quality of the folded fraction of the respective receptor.

Taken together, the findings show that the effect of the thermostabilising mutations could be partly transferred to the human 1AR. Yields of ligand binding competent receptor appear to be some 7 to 10-fold lower than for the turkey thermostabilised 1AR but should be still sufficient to perform lipid dependent ligand binding studies like the one described earlier in this thesis (see 4.3.1). For the stabilised human 1AR, fractions of ligand binding competent receptor appear to be similar to the non-stabilised turkey 1AR and in the range of ca. 1%. Additionally, soluble yields in L-CF synthesis were found to be relatively low. Thus, further optimisation might be necessary to enhance both quantity and quality of the CF-synthesis of non-stabilised human 1AR.

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