Abstract
Death-associated protein 1 (DAP1) is a proline-rich cytoplasmatic protein highly conserved in most eukaryotes. It has been reported to be involved in controlling cell growth and migration, autophagy and apoptosis. The presence of human DAP1 is associated to a favourable prognosis in different types of cancer. Here we describe the almost complete {{^{1}}text {H}}, {{^{13}}text {C}}, and {{^{15}}text {N}} chemical shift assignments of the human DAP1. The limited spectral dispersion, mainly in the {{^{1}}text {H}{^{text{N}}}} region, and the lack of defined secondary structure elements, predicted based on chemical shifts, identifies human DAP1 as an intrinsically disordered protein (IDP). This work lays the foundation for further structural investigations, dynamic studies, mapping of potential interaction partners or drug screening and development.
Highlights
The human Death-associated protein 1 (DAP1) is a member of the DAP family (DAP1-5)—originally identified as a diverse group of proteins that constitute biochemical pathways leading to apoptosis (Levy-Strumpf and Kimchi 1998)
DAP1 is rapidly activated by dephosphorylation upon inactivation of mTOR, so that the suppressive influence of dephosphorylated DAP1 acts as an antagonist to the autophagic flux (Koren et al 2010a, b)
Human bos mus rattus sus pan Displaying a general regulatory effect on cellular growth DAP1 seems to have an inhibitory effect on cell migration, autophagy and apoptosis (Koren et al 2010b; Wazir et al 2012; Xia et al 2017; Yahiro et al 2014)
Summary
The full-length human DAP1 gene, codon optimized for expression in E. coli, was ordered from Thermo Fischer Scientific (Germany) and subcloned with NdeI and XhoI restriction enzymes into a pET28a expression vector, providing an N-terminal His tag. For human DAP1 protein purification the frozen cells were resuspended in buffer (50 mM Na2HPO4 , pH 8, 300 mM NaCl, 10 mM imidazole), lysed with sonification and centrifuged at 10,000×g for 15 min. Purified human DAP1 was eluted with 0.25 M imidazole and subsequently further purified on a 16/60 HiLoad S75 size exclusion chromatography column (GE Healthcare) with 10 mM Na2HPO4 , pH 6.5, 150 mM NaCl. The fractions containing human DAP1 were pooled together and concentrated. We want to mention that the used construct has a thrombin cleavage site between the N-terminal His tag and the native human DAP1 sequence. No further thrombin cleavage site is predicted for the native human DAP1, the addition of thrombin results in the cleavage of the N-terminal His tag and in a construct shortened by 9 amino acids at the C-terminus (cleavage after R93). All NMR experiments for 1H , 15N and 13C chemical shift assignments were acquired at 10 °C in 10 mM Na2HPO4 , Backbone and nearly complete side-chain chemical shift assignments of the human
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