d2127b65f325485baeed44fa3605bbd1 pmc_33809594.pdf f2d60603c14a34f2286388b59f530609b6669846 pmc_33809594.pdf 8d33913406e2c4771a706316753a275a168b767839cca2807244e35e47ea6dad pmc_33809594.pdf Title: Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies Subject: 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 Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, 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. Keywords: inclusion bodies; affinity chromatography; dynamic light scattering; the center of spectral mass; circular dichroism; protein conformers Author: Jose Vicente Carratalá, Laia Gifre-Renom, Ramon Roca-Pinilla, Antonio Villaverde, Anna Arís, Elena Garcia-Fruitós, Julieta María Sánchez and Neus Ferrer-Miralles Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Mar 16 12:01:46 2021 CET ModDate: Tue Mar 16 12:13:47 2021 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1748135 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IVKGLO+URWPalladioL-Ital Type 1 Custom yes yes no 10 0 MKVDII+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 THNLYS+URWPalladioL-Roma Type 1 Custom yes yes no 19 0 DHJHME+EURM10 Type 1 Builtin yes yes no 23 0 PRJKGQ+VnURWPalladioL-Bold Type 1 Custom yes yes no 27 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 36 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 39 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 61 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 65 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 69 0 CBNMGP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 90 0 CBNMHB+PalatinoLinotype TrueType WinAnsi yes yes no 93 0 CBNMID+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 96 0 CBNMGP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 108 0 CBNMHB+PalatinoLinotype TrueType WinAnsi yes yes no 111 0 CBNMID+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 114 0 QMDDXF+CMSY10 Type 1 Builtin yes yes no 122 0 XHAQEC+CMBSY10 Type 1 Builtin yes yes no 126 0 CBNMGP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 133 0 CBNMHB+PalatinoLinotype TrueType WinAnsi yes yes no 136 0 CBNMID+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 139 0 CBOBCI+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 142 0 CBNMGP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 155 0 CBNMHB+PalatinoLinotype TrueType WinAnsi yes yes no 158 0 CBNMID+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 161 0 EIMZQL+PazoMath Type 1 Builtin yes yes no 171 0 FKPFBV+PazoMath-Italic Type 1 Builtin yes yes no 175 0 JHYTSG+CMR10 Type 1 Builtin yes yes no 179 0 YHLZZM+VnURWPalladioL Type 1 Custom yes yes no 186 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:24:05 CEST RepresentationInformation: pmc_33809594.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:09:35 CEST Size: 1748135 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 414 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results and Discussion Destination: section.2 Children: Item: Title: Protein Production of Soluble MMP-9 in IBs Destination: subsection.2.1 Item: Title: Solubilization of Recombinant MMP-9 from IBs Destination: subsection.2.2 Item: Title: Purification of Recombinant MMP-9 by Affinity Chromatography Destination: subsection.2.3 Item: Title: Activity of the MMP-9 Protein Peaks of L. Lactis and Clearcoli Obtained from IBs Destination: subsection.2.4 Item: Title: Physicochemical MMP-9 Properties Destination: subsection.2.5 Item: Title: Materials and Methods Destination: section.3 Children: Item: Title: Bacteria Strains and Plasmids Destination: subsection.3.1 Item: Title: Bacteria Strains and Plasmids Destination: subsection.3.2 Item: Title: Protein Purification Destination: subsection.3.3 Item: Title: Protein Detection, Yield and Purity Destination: subsection.3.4 Item: Title: MMP-9 Activity Determination by DQgelatinTM Degradation Kinetics Destination: subsection.3.5 Item: Title: Dynamic Light Scattering (DLS)s Destination: subsection.3.6 Item: Title: Determination of Intrinsic Fluorescence Destination: subsection.3.7 Item: Title: Circular Dichroism (CD) Destination: subsection.3.8 Item: Title: Statistical Analysis Destination: subsection.3.9 Item: Title: Conclusions Destination: section.4 Item: Title: Destination: section.A. Children: Item: Title: Bacterial Strains and Plasmids Destination: subsection.A.1 Item: Title: Protein Purification Destination: subsection.A.2 Item: Title: MMP-9 Activity Determination by DQgelatinTM Degradation Kinetics Destination: subsection.A.3 Item: Title: Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) Analysis Destination: subsection.A.4 Item: Title: References Destination: subsection.A.4 Info: Title: Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies Author: Jose Vicente Carratalá, Laia Gifre-Renom, Ramon Roca-Pinilla, Antonio Villaverde, Anna Arís, Elena Garcia-Fruitós, Julieta María Sánchez and Neus Ferrer-Miralles Subject: 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 Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, 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. 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