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Fig. 4 | BMC Medical Genetics

Fig. 4

From: A novel splice site indel alteration in the EIF2AK3 gene is responsible for the first cases of Wolcott-Rallison syndrome in Hungary

Fig. 4

Cloning and in vitro expression of the truncated EIF2AK3 protein. a and c shows that the cDNA of the kinase domain of the wild-type and altered protein was cloned and expressed using the pTriex4Neo expression vector and the E. coli Rosetta2 pLysS bacteria. The transfected microbes were cultured and induced with 1 mM IPTG for 0, 2 and 5 h and after that were centrifuged and lysed. The wild type and truncated EIF2AK3 protein was detected by Western-blotting using His-tag specific antibodies, a peroxidase-labeled conjugate and a chemiluminescent substrate (b and d). The wild type protein was expressed in the bacteria partly as a 72 kDa and partly as a 100 kDa protein after 2 h of induction, while after 5 h only the 100 kDa form was visible. The larger form represents the autophosphorylated EIF2AK3 molecule (b). In contrast, − as it is presented on d - the cDNA reverse transcribed from the variant mRNA coded a truncated EIF2AK3 protein with a molecular weight of 17.5 kDa and no signs of phosphorylation could be observed

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