Phage-displayed peptides as biosensor reagents

This study investigated the potential to utilize phage-displayed peptides as reagents in sensor applications. A library of random 12-mers displayed on phage was panned against staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. Nine SEB binding phage clones were isolated, all of which share the consensus sequence Trp His Lys at their amino terminus. Binding of several of these phage was shown to be inhibited when they were assayed in a competitive enzyme-linked immunosorbent assay (ELISA) format with synthesized peptide corresponding to the peptide-encoding region of one of the clones. Whole phage were labeled with the dye Cy5, and incorporated into fluoroimmunoassays. Labeled phage were able to detect SEB down to a concentration of 1.4 ng/well in a fluorescence-based immunoassay. When incorporated into an automated fluorescence-based sensing assay, Cy5-labeled phage bound to probes coated with SEB generated a robust signal of about 10,000 pA, vs a signal of 1,000 pA using a control fiber coated with streptavidin. These results demonstrate the potential for development of phage-based sensor reagents.     

The lung as a route for systemic delivery of therapeutic proteins and peptides

The large surface area, good vascularization, immense capacity for solute exchange and ultra-thinness of the alveolar epithelium are unique features of the lung that can facilitate systemic delivery via pulmonary administration of peptides and proteins. Physical and biochemical barriers, lack of optimal dosage forms and delivery devices limit the systemic delivery of biotherapeutic agents by inhalation. Current efforts to overcome these difficulties in order to deliver metabolic hormones (insulin, calcitonin, thyroid-stimulating hormone [TSH], follicle-stimulating hormone [FSH] and growth hormones) systemically, to induce systemic responses (immunoglobulins, cyclosporin A [CsA], recombinant-methionyl human granulocyte colony-stimulating factor [r-huG-CSF], pancreatic islet autoantigen) and to modulate other biological processes via the lung are reviewed. Safety aspects of pulmonary peptide and protein administration are also discussed.     

Amine-reactive isobaric tagging reagents: requirements for absolute quantification of proteins and peptides

Amine-reactive isobaric tagging reagents such as iTRAQ (isobaric tags for relative and absolute quantitation) have recently become increasing popular for relative protein quantification, cell expression profiling, and biomarker discovery. This is due mainly to the possibility of simultaneously identifying and quantifying multiple samples. The principles of iTRAQ may also be applied to absolute protein quantification with the use of synthetic peptides as standards. The prerequisites that must be fulfilled to perform absolute quantification of proteins by iTRAQ have been investigated and are described here. Three samples of somatropin were quantified using iTRAQ and synthetic peptides as standards, corresponding to a portion of the protein sequence. The results were compared with those obtained by quantification of the same protein solutions using double exact matching isotope dilution mass spectrometry (IDMS). To obtain reliable results, the appropriate standard peptides needed to be selected carefully and enzymatic digestion needed to be optimized to ensure complete release of the peptides from the protein. The kinetics and efficiency of the iTRAQ derivatization reaction of the standard peptides and digested proteins with isobaric tagging reagents were studied using a mixture of seven synthetic peptides and their corresponding labeled peptides. The implications of incomplete derivatization are also presented.     

Aptamers as therapeutic and diagnostic agents

Aptamers are oligonucleotides derived from an in vitro evolution process called SELEX. Aptamers have been evolved to bind proteins which are associated with a number of disease states. Using this method, many powerful antagonists of such proteins have been found. In order for these antagonists to work in animal models of disease and in humans, it is necessary to modify the aptamers. First of all, sugar modifications of nucleoside triphosphates are necessary to render the resulting aptamers resistant to nucleases found in serum. Changing the 2'OH groups of ribose to 2'F or 2'NH2 groups yields aptamers which are long lived in blood. The relatively low molecular weight of aptamers (8000-12000) leads to rapid clearance from the blood. Aptamers can be kept in the circulation from hours to days by conjugating them to higher molecular weight vehicles. When modified, conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target proteins. A new approach to diagnostics is also described. Aptamer arrays on solid surfaces will become available rapidly because the SELEX protocol has been successfully automated. The use of photo-cross-linkable aptamers will allow the covalent attachment of aptamers to their cognate proteins, with very low backgrounds from other proteins in body fluids. Finally, protein staining with any reagent which distinguishes functional groups of amino acids from those of nucleic acids (and the solid support) will give a direct readout of proteins on the solid support.     

Enzymes as reagents in the synthesis of peptides

The use of enzymes to catalyse peptide bond formation and for manipulating blocking groups during peptide synthesis is discussed. The history of solubility-controlled peptide condensations in the presence of proteolytic enzymes is traced. General techniques for obtaining improved yields of soluble condensation products are outlined along with special conditions which sometimes favour the enzymatic condensations of peptide fragments. Progress in the use of enzymes to manipulate blocking groups on α-amino groups, α-carboxyl groups, and on the sidechain functional groups of amino acid residues are examined. Some anticipated developments in the use of enzymes as reagents in peptide synthesis and semisynthesis are discussed.     

Analysis of cysteine residues in peptides and proteins alkylated with volatile reagents

Summary Conditions are described for the reduction and alkylation of cysteines in peptides and proteins with volatile reagents by use of triethylphosphine as reductant, bromopropane as alkylating reagent and triethylamine as base. Alkylated samples need only be vacuum dried prior to subsequent analysis steps. Alkylated samples have been acid hydrolyzed and analyzed on an amino acid analyzer with recoveries of cysteine within 10% of the expected value. Alkylated samples have been directly applied to a sequencer membrane, dried on the surface and cysteines identified by sequence analysis without additional wash steps. In addition proteins blotted onto PVDF have been alkylatedin situ and sequenced with identification of cysteines. On the analyzer and sequencer the S-propylcysteine derivative elutes at a unique position allowing for the unambiguous identification of cysteine. Cysteine residues are quantitativly alkylated under the conditions developed. The ease of this procedure allows the routine analysis of cysteine in peptides and proteins without additional, time consuming repurification or dialysis steps.Abbreviations dptu diphenylthiourea - dmptu dimethylphenylthiourea - prop-cys S-propylcysteine     

Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications

Affibody molecules are a class of engineered affinity proteins with proven potential for therapeutic, diagnostic and biotechnological applications. Affibody molecules are small (6.5 kDa) single domain proteins that can be isolated for high affinity and specificity to any given protein target. Fifteen years after its discovery, the Affibody technology is gaining use in many groups as a tool for creating molecular specificity wherever a small, engineering compatible tool is warranted. Here we summarize recent results using this technology, propose an Affibody nomenclature and give an overview of different HER2-specific Affibody molecules. Cumulative evidence suggests that the three helical scaffold domain used as basis for these molecules is highly suited to create a molecular affinity handle for vastly different applications.     

Synthetic peptides as models for intrinsic membrane proteins

There are many ways in which lipids can modulate the activity of membrane proteins. Simply a change in hydrophobic thickness of the lipid bilayer, for example, already can have various consequences for membrane protein organization and hence for activity. By using synthetic transmembrane peptides, it could be established that these consequences include peptide oligomerization, tilt of transmembrane segments, and reorientation of side chains, depending on the specific properties of the peptides and lipids used. The results illustrate the potential of the use of synthetic model peptides to establish general principles that govern interactions between membrane proteins and surrounding lipids.     

Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules

The use of antibodies in medicine and research depends on their specificity and affinity in the recogniton and binding of individual molecules. However, these applications are limited to the extracellular targets. Advances in antibody engineering has allowed the manipulation of the antibody segments containing the antigen-binding regions and generation of small fragments that can be stably expressed in cells. These entities are called intracellular antibodies or intrabodies and have being successfully applied, mainly in the scFv format, to inhibit the function of intracellular target proteins in specific cellular compartments. As new techniques to select and isolate intrabody fragments have been developed, intrabodies are beginning to be used to interfere with the function of a greater number of relevant disease targets. Just as monoclonal antibodies are opening a new era in human therapeutics, intrabodies promise a new prospective for antibody tools for therapy and research. Their varied mode of action gives intrabodies great potential in different approaches in the treatment of human diseases, as well as in the area of functional genomics for characterisation of novel gene products and subsequent validation as potential drug targets. While techniques for identifying functional intrabodies have improved, there are still many significant problems to be overcome before intrabodies can actually be used in treatment of diseases such as cancer, AIDS or neuro-degenerative disorders.     

Recombinant protein purification using complementary peptides as affinity tags

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.     

Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies

Short cationic amphiphilic peptides with antimicrobial and/or immunomodulatory activities are present in virtually every life form, as an important component of (innate) immune defenses. These host-defense peptides provide a template for two separate classes of antimicrobial drugs. Direct-acting antimicrobial host-defense peptides can be rapid-acting and potent, and possess an unusually broad spectrum of activity; consequently, they have prospects as new antibiotics, although clinical trials to date have shown efficacy only as topical agents. But for these compounds to fulfill their therapeutic promise and overcome clinical setbacks, further work is needed to understand their mechanisms of action and reduce the potential for unwanted toxicity, to make them more resistant to protease degradation and improve serum half-life, as well as to devise means of manufacturing them on a large scale in a consistent and cost-effective manner. In contrast, the role of cationic host-defense peptides in modulating the innate immune response and boosting infection-resolving immunity while dampening potentially harmful pro-inflammatory (septic) responses gives these peptides the potential to become an entirely new therapeutic approach against bacterial infections.     

Synthetic peptides derived from SARS coronavirus S protein with diagnostic and therapeutic potential

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important viral structural protein. Based on bioinformatics analysis, 10 antigenic peptides derived from the S protein sequence were selected and synthesized. The antigenicity and immunoreactivity of all the peptides were tested in vivo and in vitro. Four peptides (P6, P8, P9 and P10) which contain B cell epitopes of the S protein were identified, and P8 peptide was confirmed in vivo to have a potential in serological diagnosis. By using a syncytia formation model, we tested the neutralization ability of all 10 peptides and their corresponding antibodies. It is interesting to find that P8 and P9 peptides inhibited syncytia formation, suggesting that the P8 and P9 spanning regions may provide a good target for anti-SARS-CoV drug design. Our data suggest that we have identified peptides derived from the S protein of SARS-CoV, which are useful for SARS treatment and diagnosis.     

S100 proteins as therapeutic targets

The human genome codes for 21 S100 protein family members, which exhibit cell- and tissue-specific expression patterns. Despite sharing a high degree of sequence and structural similarity, the S100 proteins bind a diverse range of protein targets and contribute to a broad array of intracellular and extracellular functions. Consequently, the S100 proteins regulate multiple cellular processes such as proliferation, migration and/or invasion, and differentiation, and play important roles in a variety of cancers, autoimmune diseases, and chronic inflammatory disorders. This review focuses on the development of S100 neutralizing antibodies and small molecule inhibitors and their potential therapeutic use in controlling disease progression and severity.     

Macrocyclic complexes of scandium radionuclides as precursors for diagnostic and therapeutic radiopharmaceuticals

The aim of this study was to evaluate new ligands which can be applied for labeling biomolecules with scandium radionuclides. Two radionuclides of scandium, ⁴⁷Sc and ⁴⁴Sc, are perspective radioisotopes for radiotherapy and diagnostic imaging. ⁴⁷Sc decays with a half-life of 3.35 days and a maximum β⁻ energy of 600 keV and could be an alternative to carrier added ¹⁷⁷Lu radionuclide for targeted radionuclide therapy. Another scandium radionuclide ⁴⁴Sc (t_1/2 = 3.92 h) is an ideal β⁺ emitter for PET diagnosis. It can be obtained as a daughter of the long-lived ⁴⁴Ti (t_1/2 = 60.4 y) from ⁴⁴Ti/⁴⁴Sc generator. For complexation of scandium radionuclides macrocyclic ligands having a cavity size similar to Sc³⁺ ionic radius were selected: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7 triacetic acid (NOTA), 1,4,7-triazacyclodecane-1,4,7 triacetic acid and 1,4,7-triazacycloundecane triacetic acid, and analogs of NOTA with 10, 11 and 12 atoms of the carbon in the ring. Our results have shown that from the studied macrocyclic ligands studied DOTA is most efficient for binding scandium radionuclides ⁴⁴Sc and ⁴⁷Sc to biomolecules. The determined stability constant of Sc-DOTA complex logK = 27.0 is comparable with stability constants for Y³⁺ and heaviest lanthanides but is higher than those for In³⁺ and Ga³⁺. Also ⁴⁶Sc-DOTATATE conjugate exhibits high stability in-vitro studies. The ¹³C NMR studies have shown that Sc-DOTA like Lu-DOTA forms in solution complexes with eight-coordination geometry. The lipophilicity of Sc-DOTATATE is nearly identical to that of Lu-DOTATATE, which suggests similar receptor affinity of both radioconjugates.     

Natriuretic peptides as prognostic and diagnostic markers in Chagas' disease

Atrial natriuretic factor (ANF) is a hormone secreted predominantly from atrial myocardium in response to changes in wall tension. Chagas' disease is caused by the parasite Trypanosom cruzi (T. cruzi), the heart being one of the most affected organs, resulting in myocarditis and chronic cardiomyopathy. The inflammatory response of the myocardium may be the result of factors such as ischemia, direct parasite invasion, and autoimmune mechanisms. In this review, we discuss the current knowledge about ANF in Chagas' disease and describe our findings in studying: (1) the development of chagasic cardiomyophathy in T. cruzi-infected rats and its relationship with plasma ANF levels; (2) the evolution of plasma ANF in chagasic patients in different stages (asymptomatic, with conduction defects and with chronic heart failure [CHF]); and (3) the possible usefulness of plasma ANF as a prognostic factor of development of myocardial compromise and survival. In rats, the elevated ANF levels found could mirror the inflammatory response of myocardial cells to acute T. cruzi infection and of progressive failure of cardiac function in the chronic infection. In patients, plasma ANF could be a sensitive marker capable of detecting gradual impairments in cardiac function and poor survival in CHF patients and of myocardiopathy development in the asymptomatic state.     

Recombinant targeted proteins for biotherapy

In an effort to improve existing biotherapies, researchers have used recombinant techniques to alter the structure of toxins, monoclonal antibodies, and other receptor and effector molecules. Experimental research has demonstrated that the extent of problems such as nonspecific toxicity and rapid clearance by the immune system are not as great with genetically engineered toxins as opposed to native toxins. Fusion proteins, which combine portions of toxins, antibodies, or various effector molecules, exhibit the preferred biologic properties of their constituents. Unlike their murine counterparts, chimeric antibodies have the ability to invoke cell-mediated immunity and are less immunogenic to humans. Because they display different antigen-binding specificities on the same molecule, hybrid hybridomas are a potential means of juxtaposing effector cells or toxins to tumor cells. These and other positive features of recombinant proteins offer a decided advantage over previous biotherapeutic agents, and these molecules are expected to find application in the treatment of cancer, the acquired immunodeficiency syndrome, and autoimmune diseases.