Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-02-22 DOI:10.1016/j.pep.2024.106446

Peter H. Frank, Min Hong, Brianna Higgins, Shelley Perkins, Troy Taylor, Vanessa E. Wall, Matthew Drew, Timothy Waybright, William Gillette, Dominic Esposito, Simon Messing

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引用次数: 0

Abstract

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20–30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.

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将重组细菌碱性磷酸酶用于小 GTP 酶的核苷酸交换。

小 GTP 酶鼠肉瘤病毒蛋白（RAS）是细胞生长的关键调节因子，与 20%-30% 的癌症有关。RAS 通过 GTP（活性）和 GDP（非活性）的交换在其活性状态和非活性状态之间切换。因此，要研究活性蛋白，它需要与不可水解的 GTP 类似物进行核苷酸交换。与琼脂糖珠结合的小牛肠道碱性磷酸酶（CIP-琼脂糖）经常用于核苷酸交换方案，用不可水解的类似物取代 GDP。由于大流行病的供应问题和产品短缺，我们发现需要一种替代品来替代这种市售产品。在这里，我们描述了如何生成一种带有与琼脂糖珠结合的亲和性标签的细菌碱性磷酸酶（BAP）。这种 BAP 能完全交换样品中的核苷酸，从而证明了使用普通实验室设备就能替代市售产品。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.