Advantage of leaky expression, acid solubilization and CHAPS in the production of cost-effective bone morphogenetic Protein-2

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2025-01-11 DOI:10.1016/j.pep.2025.106662

Nitika Patwa, Hirah Amir, Shashank Deep

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Abstract

The aim of this study was to purify BMP-2 in an easy and time-efficient way. We have developed a new method in which BMP-2 is produced through leaky expression in E. coli BL21 (DE3) cells as inclusion bodies, eliminating the need for inducer Isopropyl β-D-1-thiogalactopyranoside (IPTG). Inclusion bodies were solubilized by the acid denaturation method. Several refolding agents, along with a reducing and oxidizing environment, were tried to produce a correctly folded dimer, which is the biologically active form of BMP-2. CHAPS was found to be the most effective refolding agent at a concentration of 20 mM. The activity of the purified protein was confirmed by alkaline phosphatase assay and calcium deposition assay on C2C12 cells and native PAGE analysis was done to check binary complex formation upon binding between BMP-2 and ALK-3 receptor. These results demonstrate that the synthesized BMP-2 protein is biologically active and has potential clinical applications.

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漏表达、酸溶解和CHAPS在高效骨形态发生蛋白-2生产中的优势。

本研究的目的是用一种简单、省时的方法纯化BMP-2。我们开发了一种新的方法，该方法通过在大肠杆菌BL21 （DE3）细胞中作为包涵体漏表达产生BMP-2，从而消除了对诱导剂异丙基β- d -1-硫代半乳糖苷（IPTG）的需要。采用酸变性法对包裹体进行溶解。几种再折叠剂，以及还原和氧化环境，试图产生正确折叠的二聚体，这是BMP-2的生物活性形式。在浓度为20 mM时，CHAPS是最有效的再折叠剂。纯化蛋白的活性通过C2C12细胞碱性磷酸酶实验和钙沉积实验证实，并通过天然PAGE分析检查BMP-2和ALK-3受体结合后形成的二元复合物。这些结果表明，合成的BMP-2蛋白具有生物活性，具有潜在的临床应用价值。

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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.