Agrin does not inhibit MMPs.

<p>Comparison of inhibitory action of miniagrin, NtA-Fs and TIMP-1 for: (A) MMP-2 (1 nM), MMP-7 (5.6 nM) and MMP-14 (4 nM) and MMP-13 (10 nM) with 50 nM TIMP-1, 100 nM Nta-FS and 1 µM miniagrin; (B) MMP-12 (1 nM) with a range of NtA-FS concentrations, 1 µM miniagrin and 50 nM TIMP-1; Values are expressed as a percentage of the uninhibited MMP activity +/− standard deviation. (C) Superimposition of NtA (Cα-backbone in pink) and TIMP-1 (Cα-backbone in steelblue) projected into the active site cleft of MMP-3 (pdb-code 1uea). Essential elements of MMP are highlighted in different color schemes (sIII-v, hB and both histidine fingers). The key disulfide bridges of TIMP-1 (Cys¹–Cys⁷⁰ and Cys³–Cys⁹⁹) and NtA (Cys²–⁷⁴) are labeled accordingly. Structural (Zn^s) and catalytic zinc (Zn^c) ions are shown as red spheres. The N-terminal sequences for NtA (in pink) and TIMP-1 (in black) reveal the missing Cys in position 1.</p

MMP-12 cleavage abolishes agrins capability to bind to laminin.

<p>(A) Dose-dependency of binding of miniagrin to laminin-coated plates indicates a specific interaction between full-length miniagrin with immobilized laminin. This interaction is abolished by MMP-12 processing of miniagrin. (B) The dose-dependent binding of the isolated NtA-FS domain to laminin is abolished by MMP-12 cleavage. Incubation with Prinomastat confirms that the MMP-12 does not process the immobilized laminin to reduce NtA interaction. Detection of the polyhistidine tag on the miniagrin and NtA-Fs was carried out with a mouse anti-his antibody, anti-mouse secondary antibody conjugated to alkaline phosphatase and alkaline phosphatase substrate. Colour development was detected at 405 nm.</p

Processing of miniagrin and NtA-Fs by matrix metalloproteases.

<p>SDS-PAGE analysis of (A) Miniagrin and (B) NtA-Fs after incubation for 18 h at 37°C alone, or with a 10∶1 molar ratio with MMP-1, 2, 7, 8, 9, 12, 13 or 14 (+). Each MMP was also incubated alone (−). Products were analysed by (A) 9% Tris-glycine (upper panel) and 15% Tris-tricine (lower panel) and (B) 15% Tris-tricine SDS-PAGE with silver staining. Following transfer to PVDF, excised products were subjected to Edman sequencing to determine cleavage sites. (C) Topology scheme of miniagrin with MMP target sites highlighted. Miniagrin is a mosaic protein composed of the following domains: NtA, N-terminal Agrin; Fs, follistatin-like; EGF, EGF–like domains 1–4 and G, globular domains 1–3. Sites A to E correspond to sites detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043669#pone-0043669-t001" target="_blank">Table 1</a>.</p

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

<div><p>Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.</p></div

Enhanced expression of eGFP.

<p>HEK293 cells were transiently transfected with eGFP alone (basic) or fused to different expression tags. (A) All proteins contained a signal peptide sequence for secretion. The eGFP signals were visualized by fluorescence microscopy. (B) To quantify the secreted eGFP, the supernatants from the HEK293 cells were excited with a laser at 488 nm and the emission signal was detected at 509 nm (top). Densitometric analysis of secreted eGFP from HEK293 cells by western blot analysis using a Strep-Tactin®-HRP conjugate to detect Strep II tagged eGFP fusion proteins (bottom). (C) Without signal peptide, eGFP was transiently expressed intracellularly with or without fused MBP. eGFP signals were visualized by fluorescence microscopy. (a.u.: arbitrary unit).</p

Biophysical analysis of MBP as well as MBP fusion proteins and cell attachment studies.

<p>(A) Light scattering profile obtained for a 1.80 mg/mL solution of MBP (dotted line) and for a 3.28 mg/mL solution of MBP-netrin-4 delta (solid line) in the TBS buffer display the respective hydrodynamic radius. (B) Concentration dependence of the hydrodynamic radius of MBP (dotted line) and MBP-netrin-4 delta (solid line), respectively, deduced from the peaks of DLS profiles. (C-D) B16-F1 cells were allowed to adhere to surface coated with MBP-netrin-4 delta. (C) Dependence of cell attachment on the coating concentration of MBP-netrin-4 delta. (D) Cell attachment at E₅₀ coating concentration (MBP-netrin-4 delta) for different netrin-4 delta proteins (with and without tag).</p

Global application of the MBP tag as an expression enhancing tag.

<p>(A) Protein expression levels in HEK293 cells of intracellular proteins (HSP90α and Gli1) with and without MBP. (B) Western blot analysis of transmembrane proteins (MEGF9 and MMP14) cloned into the basic and the MBP vector system. (C) Comparison of the expression levels of netrin-4 delta in the basic and the MBP vector transfected into COS-7, CHO-K1, and HEK293 cells. (D) The N-terminal laminin β2LN-LEa1-4 fragment and the respective R246Q mutant version causing congenital nephrotic syndrome were expressed and western blot analysis was performed.</p

Recombinant human OAS1 adopts a globular fold.

<p>(<b>A</b>) Sedimentation velocity (SV) distribution analysis in terms of <i>c</i>(S) at 0.4 mg/mL. In-set is the resultant concentration dependence of the SV distribution. (<b>B</b>) Concentration dependence of hydrodynamic radius obtained from DLS measurements. (<b>C</b>) The pair distribution function versus particle radius obtained from the GNOM analysis. In-set is the merged scattering data obtained from multiple concentrations. (<b>D</b>) Superimposition of the human OAS1 (PDB 4IG8) high-resolution structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092545#pone.0092545-Donovan1" target="_blank">[26]</a> on the <i>ab initio</i> model generated using DAMMIF on the data obtained from SAXS experiments on human OAS1.</p

Experimental and predicted hydrodynamic parameters of OAS1 and SLI/II/III (error shown in parentheses).

a<p>experimentally determined from DLS data.</p>b<p>from AUC-SV data.</p>c<p>from SAXS data.</p>d<p>the <i>r_G</i> values for R195E and K199E are 2.43 (0.11) nm and 2.40 (0.13) nm respectively.</p>e<p>the <i>D_max</i> values for R195E and K199E are 6.9 nm and 7.0 nm respectively.</p>f<p>based on homology with high-resolution structure of human OAS1.</p

The WNV SLI/II/III forms a direct interaction with human OAS1.

<p>(<b>A</b>) EMSA for OAS1 (100 nM) binding to the SLI/II/III under non-denaturing conditions. (<b>B</b>) EMSA for OAS1 (100 nM) binding to SLI+II under non-denaturing conditions. (<b>C</b>) Non-denaturing gel electrophoresis of SLI/II/III truncations (100 nM) in the presence or absence of OAS1 (400 nM). In all cases, 8% native TBE gels were used and stained with Sybr Gold (Invitrogen, USA) to visualize RNA-containing species.</p