Coexpression of nuclear receptor partners increases their solubility and biological activities
Coexpression of nuclear receptor partners increases their solubility and biological activities
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DOI：
10.1073/pnas.94.6.2278
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发表时间：
1997-03-18
影响因子：
11.1
通讯作者：
Evans, RM
中科院分区：
文献类型：
--
作者：
Li, C;Schwabe, JWR;Evans, RM
类维甲酸受体（retinoic acid receptor， RAR）和类维甲酸- x受体（retinoic - x receptor， RXR）两种核激素受体介导类维甲酸的生物活性。RXR（和它的昆虫同源超囊体）是许多其他核受体的常见异二聚体伴侣，包括昆虫蜕皮激素受体。作为核受体功能分析的一部分，我们注意到，尽管RXR在大肠杆菌中可以很容易地表达以产生可溶性蛋白，但它的许多异二聚体伴侣却不能。例如，RAR的过表达主要导致包涵体中残余的可溶性成分无法与RXR或配体有效地相互作用，其他RXR/超聚体伴侣也出现了类似的结果。我们设计了一种新的双顺反子载体，在同一细菌细胞中共表达RXR及其伴侣配体结合域，这导致可溶性异源二聚体的产量急剧增加，并且明显稳定。使用纯化的RXR- rar异源二聚体进行激素结合研究表明，这两种成分的配体结合能力增加了5到10倍，导致几乎完全的功能。基于这些研究，我们发现细菌表达的受体可以以三种不同的状态之一存在：不溶性，可溶但不能结合配体，或可溶，具有完全的配体结合能力。这些结果表明，共表达可能代表了受体复合物生物物理和结构分析的一般策略。
The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). RXR (and its insect homologue ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor. As part of a continuing analysis of nuclear receptor function, we noticed that, whereas RXR can be readily expressed in Escherichia coli to produce soluble protein, many of its heterodimeric partners cannot, For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently, Similar results are seen with other RXR/ultraspiracle partners, To overcome these problems, we designed a novel double cistronic vector to coexpress RXR and its partner ligand-binding domains in the same bacterial cell, This resulted in a dramatic increase in production of soluble and apparently stable heterodimer, Hormone-binding studies using the purified RXR-RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality. Based on these studies we find that bacterially expressed receptors can exist in one of three distinct states: insoluble, soluble but unable to bind ligand, or soluble with full ligand-binding capacity. These results suggest that coexpression may represent a general strategy for biophysical and structural analysis of receptor complexes.