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

• Published in: International journal of molecular sciences Vol. 22; no. 6; p. 3020
• Main Authors: Carratalá, Jose Vicente, Gifre-Renom, Laia, Roca-Pinilla, Ramon, Villaverde, Antonio, Arís, Anna, Garcia-Fruitós, Elena, Sánchez, Julieta María, Ferrer-Miralles, Neus
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 16.03.2021; MDPI
• Subjects: Affinity chromatography; Animals; Bacteria; Binding sites; Biological activity; Biological properties; Cattle; Chromatography; Chromatography, Affinity; Circular Dichroism; Dichroism; Dynamic Light Scattering; Efficiency; Escherichia coli - metabolism; Gelatinase B; Inclusion bodies; Inclusion Bodies - metabolism; Industrial engineering; Lactobacillus - metabolism; Light scattering; Manufacturing engineering; Matrix metalloproteinase; Matrix Metalloproteinase 9 - chemistry; Matrix Metalloproteinase 9 - isolation & purification; Matrix metalloproteinases; Metalloproteinase; Microorganisms; Molecular weight; Photon correlation spectroscopy; Physicochemical properties; Protein Conformation; protein conformers; Protein purification; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Solubility; Spectrometry, Fluorescence; Temperature; the center of spectral mass; Tryptophan - chemistry; Workflow; affinity chromatography; dynamic light scattering; protein conformers; inclusion bodies; the center of spectral mass; circular dichroism
• ISSN: 1422-0067; 1661-6596; 1422-0067
• 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.