Engineering of Recombinant Fortilin for Structure Activity Studies

Mapping Intimacies ◽

10.18122/td.1861.boisestate ◽

2021 ◽

Author(s):

Maranda S. Cantrell

Keyword(s):

Amino Acid ◽

Nmr Spectroscopy ◽

Drug Development ◽

Calcium Binding ◽

Rational Design ◽

Ldl Cholesterol ◽

Recombinant Expression ◽

High Yield ◽

Affinity Tag ◽

Binding Studies

Cardiovascular disease (CVD) is the leading cause of death worldwide affecting approximately 40% of all adults over the age of 20 and is responsible for an economic burden upwards of $3 billion annually. Treatments for CVD are limited to either hypertension medication to treat symptoms, and/or statin-based drugs to reduce low-density lipoprotein (LDL) cholesterol formation. However, recent studies suggest that approximately 50% of patients diagnosed with CVD have normal to low LDL cholesterol levels. Therefore, a critical need exists to develop new treatments for CVD that are independent of cholesterol lowering statins. Fortilin, also known as translationally controlled tumor protein (TCTP), is a 19kDa, 172 amino acid cytosolic protein ubiquitously expressed in all cell types, at all stages of life. Fortilin overexpression in arterial walls has been shown to propagate atherosclerotic plaque formation, a major component of CVD. Fortilin is therefore a promising target for the rational design of drugs to prevent formation of new plaques. For a structure-based drug development process, recombinant protein is required to characterize potential protein-drug interactions. Recombinant expression and purification of fortilin has proven to be effectual, provided any affinity tag used for purification is not cleaved. Here, we designed several constructs of recombinant fortilin fusion protein and were the first to successfully cleave the affinity tag, making structural activity studies more meaningful due to greater semblance to the native protein. Our new construct, GGS-fortilin, can be produced in high yield with greater than 85% tag cleavage, but still contains the three amino acid linkers at the N-terminus. To determine whether these three amino acids, Gly-Gly-Ser interfered with small molecule inhibitor (SMI) binding, loop constructs were designed, wherein the affinity tag, a Strep-Tactin peptide with the sequence WSHPQFEK, was placed at either the C terminal side of Arg37, Ser46 or Gly56 within the flexible loop of fortilin’s highly conserved structure and activity validated via calcium titration. Structural analysis and SMI binding studies were performed for the loop constructs using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. NMR spectroscopy is a dynamic tool used in structure-based drug development to characterize the structure of recombinantly expressed protein and to determine binding sites for SMIs. The results presented here portend to the development of novel fortilin constructs and structural studies with SMIs. Structural integrity was validated for the loop constructs using CD and NMR, and activity validated with an NMR calcium binding assay. Binding studies were also attempted using differential scanning fluorimetry (DSF) and NMR.

Download Full-text

Ten-Vertex Polyhedral Monocarbaborane Chemistry. The Novel High-Yield Formation of the Ten-Vertex closo Compound [6-(PMe2Ph)-closo-1-CB9H9] from the Thermolysis of [8,8-(PMe2Ph)2-nido-8,7-PtCB9H11]

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19932924 ◽

1993 ◽

Vol 58 (12) ◽

pp. 2924-2935 ◽

Cited By ~ 8

Author(s):

Jane H. Jones ◽

Bohumil Štíbr ◽

John D. Kennedy ◽

Mark Thornton-Pett

Keyword(s):

Nmr Spectroscopy ◽

Diffraction Analysis ◽

High Yield ◽

Monoclinic Space Group ◽

The Novel ◽

X Ray Diffraction ◽

Metal Centre ◽

X Ray ◽

Yield Formation ◽

Boiling Toluene

Thermolysis of [8,8-(PMe2Ph)2-nido-8,7-PtCB9H11] in boiling toluene solution results in an elimination of the platinum centre and cluster closure to give the ten-vertex closo species [6-(PMe2Ph)-closo-1-CB9H9] in 85% yield as a colourles air stable solid. The product is characterized by NMR spectroscopy and single-crystal X-ray diffraction analysis. Crystals (from hexane-dichloromethane) are monoclinic, space group P21/c, with a = 903.20(9), b = 1 481.86(11), c = 2 320.0(2) pm, β = 97.860(7)° and Z = 8, and the structure has been refined to R(Rw) = 0.045(0.051) for 3 281 observed reflections with Fo > 2.0σ(Fo). The clean high-yield elimination of a metal centre from a polyhedral metallaborane or metallaheteroborane species is very rare.

Download Full-text

A single amino acid substitution flanking the fourth calcium binding domain of alpha IIb prevents maturation of the alpha IIb beta 3 integrin complex.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)41800-x ◽

1994 ◽

Vol 269 (6) ◽

pp. 4450-4457

Author(s):

D.A. Wilcox ◽

J.L. Wautier ◽

D. Pidard ◽

P.J. Newman

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Calcium Binding ◽

Binding Domain ◽

Single Amino Acid Substitution ◽

Single Amino Acid ◽

Calcium Binding Domain

Download Full-text

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

International Journal of Molecular Sciences ◽

10.3390/ijms22147362 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7362

Author(s):

Amina Ben Abla ◽

Guilhem Boeuf ◽

Ahmed Elmarjou ◽

Cyrine Dridi ◽

Florence Poirier ◽

...

Keyword(s):

Specific Binding ◽

Cellular Adhesion ◽

High Yield ◽

Adhesion Assay ◽

Cell Binding ◽

Affinity Tag ◽

Binding Domains ◽

Rational Development ◽

Fibronectin Type Iii ◽

Arginine Glycine Aspartic Acid

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.

Download Full-text

Generation of Monoclonal Antibodies for Sensitive Detection of Pro-Inflammatory Protein S100A9

Applied Sciences ◽

10.3390/app11104659 ◽

2021 ◽

Vol 11 (10) ◽

pp. 4659

Author(s):

Eun-Jung Kim ◽

Gyu-Min Im ◽

Chang-Soo Lee ◽

Yun-Gon Kim ◽

Byoung Joon Ko ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Calcium Binding ◽

Nucleotide Sequences ◽

Antibody Fragments ◽

High Yield ◽

Antigen Binding ◽

Hybridoma Cell Culture ◽

Inflammatory Protein ◽

Inflammatory Processes ◽

Yield And Purity

The calcium-binding protein S100A9 regulates inflammatory processes and the immune response. It is overexpressed in a variety of inflammatory and oncologic conditions. In this study, we produced a recombinant human S100A9 (hS100A9) antigen with high yield and purity and used it to generate a hybridoma cell culture-based monoclonal anti-hS100A9 antibody. We selected five anti-hS100A9 antibodies from cell supernatants that showed high antigen binding efficiency and identified the nucleotide sequences of three antibodies: two with high effective concentration values and one with the lowest value. The antigen and antibody development procedures described herein are useful for producing large amounts of monoclonal antibodies against hS100A9 and other antigens of interest. The nucleotide sequences of the anti-hS100A9 monoclonal antibody revealed herein will be helpful in the generation of recombinant antibodies or antibody fragments against hS100A9.

Download Full-text

Rational design of novel amphipathic antimicrobial peptides focused on the distribution of cationic amino acid residues

MedChemComm ◽

10.1039/c9md00166b ◽

2019 ◽

Vol 10 (6) ◽

pp. 896-900 ◽

Cited By ~ 5

Author(s):

Takashi Misawa ◽

Chihiro Goto ◽

Norihito Shibata ◽

Motoharu Hirano ◽

Yutaka Kikuchi ◽

...

Keyword(s):

Antimicrobial Activity ◽

Amino Acid ◽

Human Cell ◽

Hemolytic Activity ◽

Rational Design ◽

Cell Cytotoxicity ◽

Amino Acid Residues ◽

High Antimicrobial Activity ◽

Cationic Amino Acid ◽

Helical Peptide

Amphipathic helical peptideStripeshowed high antimicrobial activity, low hemolytic activity, and low human cell cytotoxicity.

Download Full-text

HLA-DQB1 codon 57 is critical for peptide binding and recognition.

Journal of Experimental Medicine ◽

10.1084/jem.183.3.1253 ◽

1996 ◽

Vol 183 (3) ◽

pp. 1253-1258 ◽

Cited By ~ 75

Author(s):

W W Kwok ◽

M E Domeier ◽

M L Johnson ◽

G T Nepom ◽

D M Koelle

Keyword(s):

Amino Acid ◽

T Cell ◽

Cell Clone ◽

Peptide Binding ◽

Class Ii ◽

Binding Studies ◽

Peptide Complex ◽

Mhc Molecules ◽

Hla Dq ◽

T Cell Clone

The association of specific HLA-DQ alleles with autoimmunity is correlated with discrete polymorphisms in the HLA-DQ sequence that are localized within sites suitable for peptide recognition. The polymorphism at residue 57 of the DQB1 polypeptide is of particular interest since it may play a major structural role in the formation of a salt bridge structure at one end of the peptide-binding cleft of the DQ molecules. This polymorphism at residue 57 is a recurrent feature of HLA-DQ evolution, occurring in multiple distinct allelic families, which implies a functional selection for maintaining variation at this position in the class II molecule. We directly tested the amino acid polymorphism at this site as a determinant for peptide binding and for antigen-specific T cell stimulation. We found that a single Ala-->Asp amino acid 57 substitution in an HLA-DQ3.2 molecule regulated binding of an HSV-2 VP-16-derived peptide. A complementary single-residue substitution in the peptide abolished its binding to DQ3.2 and converted it to a peptide that can bind to DQ3.1 and DQ3.3 Asp-57-positive MHC molecules. These binding studies were paralleled by specific T cell recognition of the class II-peptide complex, in which the substituted peptide abolished T cell reactivity, which was directed to the DQ3.2-peptide complex, whereas the same T cell clone recognized the substituted peptide presented by DQ3.3, a class II restriction element differing from DQ3.2 only at residue 57. This structural and functional complementarity for residue 57 and a specific peptide residue identifies this interaction as a key controlling determinant of restricted recognition in HLA-DQ-specific immune response.

Download Full-text

Three distinct anti-allergic drugs, amlexanox, cromolyn and tranilast, bind to S100A12 and S100A13 of the S100 protein family

Biochemical Journal ◽

10.1042/bj3380583 ◽

1999 ◽

Vol 338 (3) ◽

pp. 583-589 ◽

Cited By ~ 36

Author(s):

Tsuyoshi SHISHIBORI ◽

Yuhta OYAMA ◽

Osamu MATSUSHITA ◽

Kayoko YAMASHITA ◽

Hiromi FURUICHI ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Calcium Binding ◽

Binding Proteins ◽

S100 Protein ◽

Reversed Phase ◽

Molecular Probes ◽

Calcium Binding Proteins ◽

Ige Mediated ◽

Human And Mouse

To investigate the roles of calcium-binding proteins in degranulation, we used three anti-allergic drugs, amlexanox, cromolyn and tranilast, which inhibit IgE-mediated degranulation of mast cells, as molecular probes in affinity chromatography. All of these drugs, which have different structures but similar function, scarcely bound to calmodulin in bovine lung extract, but bound to the same kinds of calcium-binding proteins, such as the 10-kDa proteins isolated in this study, calcyphosine and annexins I–V. The 10-kDa proteins obtained on three drug-coupled resins and on phenyl-Sepharose were analysed by reversed-phase HPLC. It was found that two characteristic 10-kDa proteins, one polar and one less polar, were bound with all three drugs, although S100A2 (S100L), of the S100 family, was bound with phenyl-Sepharose. The cDNA and deduced amino acid sequence proved our major polar protein to be identical with the calcium-binding protein in bovine amniotic fluid (CAAF1, S100A12). The cDNA and deduced amino acid sequence of the less-polar protein shared 95% homology with human and mouse S100A13. In addition, it was demonstrated that the native S100A12 and recombinant S100A12 and S100A13 bind to immobilized amlexanox. On the basis of these findings, we speculate that the three anti-allergic drugs might inhibit degranulation by binding with S100A12 and S100A13.

Download Full-text