Liposomes as formulation excipients for protein pharmaceuticals: a model protein study.
Liposomes as formulation excipients for protein pharmaceuticals: a model protein study.
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脂质体作为蛋白质药物制剂赋形剂：模型蛋白质研究。
DOI：
10.1023/a:1007561308498
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发表时间：
2000
影响因子：
3.7
通讯作者：
Straubinger,RM
中科院分区：
文献类型：
--
作者：
Balasubramanian,SV;Bruenn,J;Straubinger,RM
目的。重组DNA技术的出现使多种蛋白质和多肽的医药用途成为可能，但蛋白质的复杂结构使得它们容易受到变性、聚集和沉淀等物理不稳定性的影响。我们验证了疏水结构域的部分展开和暴露会导致物理不稳定的假设，并研究了稳定蛋白质配方的方法。以玉米黑粉菌(Ustilago Maydis)的81个氨基酸的致死毒素KP6β为模型蛋白。用圆二色谱和荧光光谱研究了KP6β的温度依赖性折叠/去折叠特性。1，8-苯胺基-1，8-萘磺酸盐(ANS)是一种可分割成疏水结构域的荧光探针，用于检测疏水结构域的暴露。随着温度的升高，近紫外镉表明KP6β的三级结构逐渐丧失，而远紫外镉表明二级结构保持不变。ANS的穿透表明，疏水结构域暴露增加。在高温(60℃)下，KP6β保留了大部分二级结构特征。然而，三级结构是无序的，表明存在部分折叠的、结构化的中间状态。脂质体结合到部分展开的结构上，阻止了聚集体的形成。部分展开导致KP6β疏水结构域暴露和聚集增加，但二级结构保持不变。脂质体与结构中间态相互作用，稳定蛋白质的聚集。这些结果提出了一种蛋白质的一般配方策略，其中部分展开的结构通过配方辅料作为分子伴侣来稳定，以避免物理上的不稳定。
Purpose. The advent of recombinant DNA technology has madepossible the pharmaceutical use of a wide range of proteins and peptides.However, the complex structure of proteins renders them susceptibleto physical instabilities such as denaturation, aggregation andprecipitation. We tested the hypothesis that partial unfolding and exposure ofhydrophobic domains leads to physical instability, and investigatedapproaches to stabilize protein formulations.Methods. KP6 β, an 81 amino acid killer toxin fromUstilago maydis,was used as a model protean. Circular dichroism and fluorescencespectroscopy were used to study the temperature dependent folding/unfolding characteristics of KP6 β. ANS (1,8 anilinonaphthalenesulfonate), a fluorescent probe that partitions into hydrophobic domains,was used to detect exposure of hydrophobic domains.Results. As the temperature was elevated, near-UV CD indicatedprogressive loss of KP6 β tertiary structure, while far-UV CD indicatedretention of secondary structure. Increasing exposure of hydrophobicdomains was observed, as indicated by the penetration of ANS. Atelevated temperatures (60°C), KP6 β conserved most secondarystructural features. However, tertiary structure was disordered, suggestingthe existence of a partially folded, structured intermediate state.Liposomes bound to partially unfolded structures and prevented theformation of aggregates.Conclusions. Partial unfolding resulted in increased exposure ofhydrophobic domains and aggregation of KP6 β, but with preservationof secondary structure. Liposomes interacted with the structuredintermediate state, stabilizing the protein against aggregation. These resultssuggest a general formulation strategy for proteins, in which partiallyunfolded structures are stabilized by formulation excipients that act asmolecular chaperones to avoid physical instability.