Expression, DNA- and amino-acid sequences of PLIN1/3 chimeric constructs

Perilipin 1 (PLIN1) represents a major structural component on the periphery of lipid droplets (LDs) and plays a central role in modulating adipocyte metabolic processes through its interaction with key proteins. Out of the five plin genes identified in mammals the gene products PLIN1, 2, 3 and 5 share a conserved N-terminal architecture which comprises a hydrophobic PAT (Perilipin/ADRP/TIP47) domain and an 11-mer repeat domain of varying length. However, PLIN1 contains a unique C-terminus compared to other members of the perilipin family, and human PLIN1-3 differ substantially in their cytoplasmic stability, which is attributed to differences in the architecture of the C-terminal 4-helix bundle. In PLIN3, the 4-helix bundle is held together by an α/β domain enhancing its stability in the cytoplasm. In contrast, the stabilising α/β domain is believed to be absent in PLIN1, thereby increasing its tendency to associate with phospholipid membranes of LDs. To obtain protein with increased stability that can be used for functional and structural studies of the interplay between PLIN1 and its interaction partners in an aqueous environment, we designed and expressed two different perilipin chimeras. Both constructs retain the N- and C-terminal domains of PLIN1 that are crucial for protein-protein interactions but contain different proportions of the more stable PLIN3 LD-interacting PAT and/or 11-mer repeat domains. The putative domain boundaries of PLIN1 were determined using different online tools for secondary-structure analysis (PSIPRED, metaPrDOS, MINNOU, Phyre2 and EMBOSS pepwheel) as well as the structural information from murine PLIN3 (PDB: 1SZI). An expression test revealed differences in distribution of the two chimeras between soluble and insoluble fractions. Particularly, the PLIN1/3 Chimera 1 construct containing the 11-mer domain, α/β domain and the 4-helix bundle of PLIN3 was enriched in the soluble fraction upon expression in E.coli. Here, we provide the DNA and amino-acid sequences of the two chimeric PLIN constructs.