Simplification of the purification of heat stable recombinant low molecular weight proteins and peptides from GST-fusion products.

The synthesis and purification of peptides of importance in the fields of research and medicine continue to be a challenging task. Chemical synthesis of oligopeptides, especially those greater than 25 amino acids, is cost prohibitive. On the other hand, several bottlenecks exist in the production of recombinant short peptides in heterologous expression hosts such as Escherichia coli (E.

Molecular insights on cytochrome c and nucleotide regulation of apoptosome function and its implication in cancer.

Cytochrome c (Cyt c) released from mitochondria interacts with Apaf-1 to form the heptameric apoptosome, which initiates the caspase cascade to execute apoptosis. Although lysine residue at 72 (K72) of Cyt c plays an important role in the Cyt c-Apaf-1 interaction, the underlying mechanism of interaction between Cyt c and Apaf-1 is still not clearly defined. Here we identified multiple lysine residues including K72, which are also known to interact with ATP, to play a key role in Cyt c-Apaf-1 interaction.

Design of a thrombin resistant human acidic fibroblast growth factor (hFGF1) variant that exhibits enhanced cell proliferation activity.

Acidic fibroblast growth factors (FGF1s) are heparin binding proteins that regulate a wide array of key cellular processes and are also candidates for promising biomedical applications. FGF1-based therapeutic applications are currently limited due to their inherent thermal instability and susceptibility to proteases. Using a wide range of biophysical and biochemical techniques, we demonstrate that reversal of charge on a well-conserved positively charged amino acid, R136, in the heparin binding pocket drastically increases the resistance to proteases, thermal stability, and cell proliferation activity of the human acidic fibroblast growth factor (hFGF1).

Molecular mechanisms of heparin-induced modulation of human interleukin 12 bioactivity.

Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration.

Probing the role of proline -135 on the structure, stability, and cell proliferation activity of human acidic fibroblast growth factor.

Human acidic fibroblast growth factor 1 (hFGF1) is a protein intricately involved in cell growth and tissue repair. In this study, we investigate the effect(s) of understanding the role of a conserved proline (P135), located in the heparin binding pocket, on the structure, stability, heparin binding affinity, and cell proliferation activity of hFGF1. Substitution of proline-135 with a positively charged lysine (P135K) resulted in partial destabilization of the protein; however, the overall structural integrity of the protein was maintained upon substitution of proline-135 with either a negative charge (P135E) or a polar amino acid (P135Q).

Identification of avian vasotocin receptor subtype-specific antagonists involved in the stress response of the chicken, Gallus gallus.

Vasotocin 1a and 1b receptors (V1aR and V1bR) have been shown to play important roles in the neuroendocrine regulation of stress responses via the anterior pituitary (AP) of birds. To identify effective subtype-specific antagonists for the chicken V1aR (cV1aR) and cV1bR, potential antagonists to the mammalian V1R were screened against the cV1aR and cV1bR 3D structural models by molecular docking analysis with determination of binding pocket/amino acid residues involved in the interaction. The antagonistic effects of the selected ligands were examined by measuring pro-opiomelanocortin (POMC) heteronuclear RNA (hnPOMC) levels following the in vitro stress administration to primary chicken AP cells.

Effect of extension of the heparin binding pocket on the structure, stability, and cell proliferation activity of the human acidic fibroblast growth factor.

Acidic human fibroblast growth factor (hFGF1) plays a key role in cell growth and proliferation. Activation of the cell surface FGF receptor is believed to involve the glycosaminoglycan, heparin. However, the exact role of heparin is a subject of considerable debate.

Stilbenoid prenyltransferases define key steps in the diversification of peanut phytoalexins.

Defense responses of peanut () to biotic and abiotic stresses include the synthesis of prenylated stilbenoids. Members of this compound class show several protective activities in human disease studies, and the list of potential therapeutic targets continues to expand. Despite their medical and biological importance, the biosynthetic pathways of prenylated stilbenoids remain to be elucidated, and the genes encoding stilbenoid-specific prenyltransferases have yet to be identified in any plant species.

Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest.

Backbone and side-chain H, N, and C resonance assignments of a novel Staphylococcal inhibitor of myeloperoxidase.

The bacterium Staphylococcus aureus produces an array of anti-inflammatory molecules that prevent the innate immune system from recognizing it as a pathogen and clearing it from the host. In the acute phase of inflammation, our immune system relies on neutrophils to clear invading bacteria. Recently, novel classes of secreted proteins from S.

Modulation of Interleukin-12 activity in the presence of heparin.

Glycosaminoglycans (GAGs), especially heparin and heparan sulfate (HS), modulate the functions of numerous cytokines. The aims of this multidisciplinary research were to characterize heparin binding to interleukin-12 (IL-12) and determine the mechanism(s) by which heparin influences IL-12 bioactivity. Heparin and HS were found to bind human IL-12 (hIL-12) with low micromolar affinity and increase hIL-12 bioactivity by more than 6-fold.

Novel Molecular Interactions of Acylcarnitines and Fatty Acids with Myoglobin.

Previous research has indicated that long-chain fatty acids can bind myoglobin (Mb) in an oxygen-dependent manner. This suggests that oxy-Mb may play an important role in fuel delivery in Mb-rich muscle fibers (e.g.

Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle.

Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs.

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms.

Production of an anti-Candida peptide via fed batch and ion exchange chromatography.

Interest in peptides as diagnostic and therapeutic materials require their manufacture via either a recombinant or synthetic route. This study examined the former, where a recombinant fusion consisting of an antifungal peptide was expressed and isolated from Escherichia coli. Fed batch fermentation with E.

Folding of Fibroblast Growth Factor 1 Is Critical for Its Nonclassical Release.

Fibroblast growth factor 1 (FGF1), a ubiquitously expressed pro-angiogenic protein that is involved in tissue repair, carcinogenesis, and maintenance of vasculature stability, is released from the cells via a stress-dependent nonclassical secretory pathway. FGF1 secretion is a result of transmembrane translocation of this protein. It correlates with the ability of FGF1 to permeabilize membranes composed of acidic phospholipids.

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates.

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome.

Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of acidic fibroblast growth factor.

Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER)-Golgi secretory pathway. FGF1 is released through a Cu(2+)-mediated nonclassical secretion pathway.

Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag.

Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration.

Controlling chitosan-based encapsulation for protein and vaccine delivery.

Chitosan-based nano/microencapsulation is under increasing investigation for the delivery of drugs, biologics and vaccines. Despite widespread interest, the literature lacks a defined methodology to control chitosan particle size and drug/protein release kinetics. In this study, the effects of precipitation-coacervation formulation parameters on chitosan particle size, protein encapsulation efficiency and protein release were investigated.

Identification of antagonists to the vasotocin receptor sub-type 4 (VT4R) involved in stress by molecular modelling and verification using anterior pituitary cells.

The vasotocin receptor family is homologous to the mammalian vasopressin G-protein coupled receptor (GPCR) family. The vasotocin receptor 2 (VT2R) and 4 (VT4R) have recently been shown to play important role(s) in the neuroendocrine regulation of stress in birds. A homology-based structural model of VT4R of the domestic chicken, Gallus gallus, was built using the sophisticated SYBYL-X suite.

Elevated Expression of indoleamine 2,3-dioxygenase (IDO) and accumulation of kynurenic acid in the pathogenesis of STZ-induced diabetic cataract in Wistar rats.

Purpose: Glycated lens proteins are capable of producing reactive oxygen species (ROS), which, in turn, can oxidize tryptophan (Trp) into kynurenines. Indoleamine 2,3-dioxygenase (IDO), which is expressed in many tissues and which is inducible by interferon-gamma (IFN-gamma), is able to oxidize Trp into kynurenines. These kynurenines can modify lens proteins and, in fact, kynurenine adducts are markedly increased in lenses with age-related nuclear cataract.