Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines

Abstract

The site-specific incorporation of cross-linkable designer amino acids into proteins is useful for covalently bonding protein complexes upon exposure to light. This technology can be used to study networks of protein-protein interactions in living cells; however, to date it has only been applicable for use with a narrow range of cell types, due to the limited availability of plasmid-based transfection protocols. In the present study, we achieved adenovirus-based expression of a variant of an archaeal pyrrolysyl-tRNA synthetase and UAG-recognising tRNA pair, which was used to incorporate unnatural amino acids into proteins at sites defined by in-frame UAG codons within genes. As such, the site-specific photo-cross-linking method is now applicable to a wide variety of mammalian cells. In addition, we repositioned the reactive substituent of a useful photo-cross-linker, Nε-(para-trifluoromethyl-diazirinyl-benzyloxycarbonyl)-l-lysine (pTmdZLys), to the meta position, which improved its availability at low concentration. Finally, we successfully applied this system to analyse the formation of a protein complex in response to a growth signal in human cancerous cells and human umbilical vein endothelial cells. This adenovirus-based system, together with the newly designed cross-linkable amino acid, will facilitate studies on molecular interactions in various cell lines of medical interest.

Cell-autonomous and redundant roles of Hey1 and HeyL in muscle stem cells: HeyL requires Hes1 to bind diverse DNA sites

“…Thus, we examined the necessity of Hes1 for HeyL because it is possible that Hes1 and HeyL work as a heterodimer (Jalali et al, 2011). First, we assayed the existence of HeyL-Hes1 heterodimers in living cells using a site-specific photo-crosslink technique (Hino et al, 2005;Kita et al, 2016) (Fig. 4A).…”

“…Site-specific incorporation of mTmdZLys into HeyL protein in living cells with an expanded genetic code was performed as described previously (Kita et al, 2016). In brief, a pOriP plasmid containing the Heyl gene with an amber nonsense (TAG) mutation in the positions shown in Fig.…”

“…For protein photocrosslinking, the cells were exposed to UV light (λ=365 nm) for 15 min. Extraction, purification, and western blot analysis of crosslinked products were performed as described previously (Kita et al, 2016). The analyses of the heterodimerization of proteins other than Hey1-Hes1were performed in a similar way.…”

A Spotlight on Viruses—Application of Click Chemistry to Visualize Virus-Cell Interactions

“…Efficient gene transfer methods using virus-derived vector systems present an alternative, also providing more flexibility regarding the cell type used. For this, several virus-derived gene transfer vectors are being explored, including vectors derived from adenoviruses [42], lentiviruses [43], AAV [44] and baculoviruses [45]. With their large coding capacity and ability to efficiently transduce different cell types (e.g., primary cells including mouse embryonic fibroblasts and rat cardiac fibroblasts, cultured neurons, as well as mouse embryonic stem cells), baculoviral vectors are a particularly attractive possibility for experiments in tissue culture with cell types which are difficult to manipulate [45].…”

Synthesis at the interface of virology and genetic code expansion

“…Additionally, to achieve optimal expression levels of the suppressor tRNA, multiple copies of its expression cassette must be encoded in the viral genome [52,53], which can be challenging due to its recombination-prone nature. So far, several viral vectors has been developed for this purpose, including lentivirus,[54] adenovirus,[55] AAV[56] and baculovirus[52,57]. The efficiency of Uaa incorporation observed with lentiviral and adenoviral systems appears to be low.…”