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.脂滴包被蛋白1（Perilipin 1, PLIN1）是脂滴（lipid droplets, LDs）外周的主要结构组分，可通过与关键蛋白的相互作用调控脂肪细胞的代谢过程，发挥核心功能。在哺乳动物中已鉴定出5种plin基因，其编码产物PLIN1、2、3和5拥有保守的N端结构架构，该架构包含一个疏水性PAT（Perilipin/ADRP/TIP47）结构域与一段长度各异的11聚体重复结构域。但与脂滴包被蛋白家族的其他成员相比，PLIN1拥有独特的C端结构域；且人类PLIN1至PLIN3的胞质稳定性差异显著，这一差异源于其C端4螺旋束的结构架构不同。在PLIN3中，4螺旋束由一个α/β结构域维系，这提升了其在胞质中的稳定性。与之相反，学界普遍认为PLIN1缺乏该具有稳定作用的α/β结构域，因此PLIN1更倾向于与脂滴的磷脂膜结合。为获得稳定性提升的蛋白，以用于水环境下PLIN1与其互作伴侣间相互作用的功能与结构研究，本研究设计并表达了两种不同的脂滴包被蛋白嵌合体。两种构建体均保留了PLIN1中对蛋白质相互作用至关重要的N端与C端结构域，但引入了不同比例的稳定性更强的、可与脂滴结合的PLIN3的PAT结构域和/或11聚体重复结构域。研究人员通过多种在线二级结构分析工具（PSIPRED、metaPrDOS、MINNOU、Phyre2及EMBOSS pepwheel），结合小鼠PLIN3的结构信息（PDB: 1SZI），确定了PLIN1的推定结构域边界。表达试验结果显示，两种嵌合体在可溶性与不可溶性组分中的分布存在差异。具体而言，包含PLIN3的11聚体结构域、α/β结构域与4螺旋束的PLIN1/3嵌合体1，在大肠杆菌（E. coli）中表达时可在可溶性组分中富集。本文提供了这两种嵌合型PLIN构建体的DNA序列与氨基酸序列。