Improved production of class I phosphatidylinositol 4,5-bisphosphate 3-kinase

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-08-20 DOI:10.1016/j.pep.2024.106582

Simon Messing, Stephanie R.T. Widmeyer, John-Paul Denson, Jennifer Mehalko, Vanessa E. Wall, Matthew Drew, Kelly Snead, Min Hong, Carissa Grose, Dominic Esposito, William Gillette

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引用次数: 0

Abstract

Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the β and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases.

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改进 I 类磷脂酰肌醇-4,5-二磷酸 3-激酶的生产。

磷脂酰肌醇-4,5-二磷酸 3-激酶（PI3K）是一个激酶家族，其活性影响着细胞基本功能所需的通路。因此，PI3K 是所有人类癌症中突变最多的基因之一，也是癌症治疗的理想靶点。在其他研究小组所做工作的基础上，我们通过改进溶解度标签、新颖的表达方式、优化纯化方案和缓冲液等多种方法，提高了蛋白质产量，生产出稳定纯净的蛋白质。通过这些方法，我们将 p110α/p85α 的产量提高了 40 倍，将 p110α 的产量提高了 3 倍。我们还用这些方案制备了 PI3K 的 β 和 δ 异构体的可比构建体。产量的增加提高了我们对 PIK3 激酶家族的下游高通量药物发现工作的效率。

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.