Expression and purification of the extracellular domain of wild-type humanRET and the dimeric oncogenic mutant C634R

Abstract

This is a repository copy of Expression and purification of the extracellular domain of wildtype humanRET and the dimeric oncogenic mutant C634R.

Unexpected structures formed by the kinase RET C634R mutant extracellular domain suggest potential oncogenic mechanisms in MEN2A

“…The expression of correctly folded and functional human RET requires the use of mammalian expression systems and we previously reported that the addition of a cleavable C-terminal Fc tag facilitates the expression of RET C634R ECD in its dimeric form, mediated by an intermolecular C630-C630 disulfide bond ( 25 ). For this study, we further optimized the expression construct (see Experimental procedures for details) and were able to isolate dimeric RET C634R ECD in high homogeneity ( Fig.…”

“…Taken together with the BN PAGE and SEC-MALS data, these results suggest that RET C634R ECD forms a complex with GDF15 and GFRAL in a stoichiometry of 4:4:4 that must exist in a different configuration from the RET WT ECD complex. Indeed, we measured that the affinity of RET C634R ECD for GDF15/GFRAL ( K d = 8.5 μM) is actually weaker than that of RET WT ECD ( K d = 3.2 μM) ( 25 ) ( Fig. S3 ).…”

“…Comparing our map to a previously published cryo-EM structure of the same complex (PDB ID: 6Q2J ) ( 16 ), we observed only minor differences, most notably extra density, consistent with the additional glycosylation at certain sites. RET ECD is heavily glycosylated, containing 11 glycosylation sites that influence the structure and function of RET ( 25 , 27 , 28 ). In our study, we used HEK293T cells to produce RET ECD bearing near-native glycans and, at this resolution, densities of the base glycans at all the glycosylation sites could be observed ( Fig.…”

“…WT RET ECD (residues 1–635) was subcloned into pcDNA3.1 vector with a Tobacco Etch Virus protease cleavage site and His 8 -Flag tags (RET WT ). Human GDF15 (residues 195–308) was cloned into the same vector with a modified N-terminal Fc tag, followed by a thrombin cleavage site, and this construct was used for cotransfection with a separate construct for the expression of modified Fc as described previously ( 25 , 40 ). The construct generation and expression procedures of RET WT , Fc-GDF15, and human GFRAL (residues 19–351) with C-terminal His 8 -twin Strep tags were the same as previously described ( 25 ).…”

“…A BLItz instrument (FortéBio) was used to measure the binding affinity between RET C634R and Fc-GDF15/GFRAL and the experimental set up was as previously described (25). Fc-GDF15 was obtained through fitting a nonlinear regression sigmoidal model using GraphPad Prism 8.…”