Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain.
Department of Genetics and Microbiology, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain.
Int J Mol Sci. 2021 Mar 16;22(6):3020. doi: 10.3390/ijms22063020.
A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories (an engineered form of ) and mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.
我们遵循了一个详细的工作流程，用于分析从蛋白质聚集体（包涵体）中获得的哺乳动物基质金属蛋白酶（MMP-9）蛋白物种的物理化学特性。MMP-9 在原核微生物细胞工厂（一种工程形式的）中重组产生，主要形成包涵体的一部分，并在非变性条件下部分回收。在通过亲和层析对溶解的 MMP-9 进行纯化后，得到了四个蛋白质峰。然而，到目前为止，尚未分离和表征构成包涵体的不同构象蛋白物种。因此，为了将分离峰的物理化学特性与其生物学活性联系起来，我们建立了一种方法学方法，包括动态光散射（DLS）、圆二色性（CD）和荧光光谱分析，证实了具有特定特征的构象亚群的分离。在蛋白质纯化过程中，通过色谱技术分离的蛋白质峰的单个物理化学特性和生物学活性的详细分析是选择最佳拟合蛋白质群体的可靠信息来源。
