Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies

• Main Authors: Jose Vicente Carratalá, Laia Gifre-Renom, Ramon Roca-Pinilla, Antonio Villaverde, Anna Arís, Elena Garcia-Fruitós, Julieta María Sánchez, Neus Ferrer-Miralles
• Format: Article
• Language: English
• Published: MDPI AG 2021-03-01
• Series: International Journal of Molecular Sciences
• Subjects: inclusion bodies; affinity chromatography; dynamic light scattering; the center of spectral mass; circular dichroism; protein conformers
• Online Access: https://www.mdpi.com/1422-0067/22/6/3020

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 <i>Clearcoli</i> (an engineered form of <i>Escherichia coli</i>) and <i>Lactococcus lactis,</i> 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.