CICS-UBI-Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, Portugal.
Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal.
Int J Mol Sci. 2021 Sep 16;22(18):10012. doi: 10.3390/ijms221810012.
The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive.
The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (m) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism.
The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A m of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein's structural stability.
A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.
STEAP1 是一种在前列腺癌中过度表达的细胞表面抗原，它有助于肿瘤的进展和侵袭。然而，STEAP1 的分子机制及其结构决定因素仍不清楚。
通过改变结合和洗脱相的离子强度来评估丁基-和辛基-琼脂糖基质对 LNCaP 裂解物的分馏能力，然后进行共免疫沉淀（Co-IP）抛光。评估了几种潜在的稳定化添加剂，并根据熔点（m）值对最佳/最差化合物进行了排序。通过圆二色性确定 STEAP1 的二级结构。
STEAP1 不能用 1.375 M（丁基）完全捕获，而干扰的异源蛋白则被强烈保留，大部分用水洗脱。这一步骤表明丁基琼脂糖对从注射粗样品中去除宿主杂质具有更高的选择性。Co-IP 允许回收纯化的 STEAP1 部分，并有助于揭示与目标潜在的生理相互作用的对应物。确定了~55°C 的 m 值，证实了 STEAP1 在纯化缓冲液中的稳定性。确定了主要的α-螺旋结构，确保了蛋白质的结构稳定性。
提供了一种从 LNCaP 细胞中成功分离人 STEAP1 的方法，避免使用去污剂来实现稳定性，即使在模拟膜环境之外也是如此。
