Protein detection by Western blot via coiled-coil interactions

We propose an approach for the detection of proteins by Western blot that takes advantage of the high-affinity interaction occurring between two de novo designed peptides, the E and K coils. As a model system, K coil-tagged epidermal growth factor (EGF) was revealed with secreted alkaline phosphatase (SeAP) tagged with E coil (SeAP-Ecoil) as well as with biotinylated E coil. In that respect, we first produced purified SeAP-Ecoil and verified its ability to interact with K coil peptides by surface plasmon resonance biosensing. We demonstrated that protein detection with Ecoil-biotin was more specific than with SeAP-Ecoil. We then showed that our approach is as sensitive as conventional detection strategies relying on nickel-nitrilotriacetic acid-horseradish peroxidase (Ni-NTA-HRP), anti-His-HRP, or anti-EGF. Altogether, our results indicate that the E/K coiled-coil system is a good alternative for protein detection by Western blot.     

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost‐effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co‐immobilized them on modified poly‐p‐hydroxybutyrate synthase (PhaC)‐inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled‐coil interaction. Enzyme‐loaded particles could be easily purified by centrifugation. Total conversion of 4'‐chloroacetophenone to (S)‐4‐chloro‐α‐methylbenzyl alcohol could be accomplished using enzyme‐loaded particles, catalytic amounts of NAD⁺ and formate as substrates for FDH. Chiral GC‐MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost‐effective alternative to coupled reactions using purified enzymes.     

Epidermal growth factor radiopharmaceuticals: 111In chelation, conjugation to a blood-brain barrier delivery vector via a biotin-polyethylene linker, pharmacokinetics, and in vivo imaging of experimental brain tumors.

Epidermal growth factor (EGF) is a potential peptide radiopharmaceutical for detection of brain tumors, because many human gliomas overexpress the EGF receptor (EGFR). The transport of EGF to the brain, however, is restricted by the blood-brain barrier (BBB). The purpose of the present study was to develop a vector-mediated brain delivery system for radiolabeled EGF. Human EGF was monobiotinylated with NHS-PEG3400-biotin, where NHS is N-hydroxysuccinimide and PEG3400 is poly(ethylene glycol) of 3400 Da molecular mass. EGF-PEG3400-biotin was radiolabeled with either 125I or 111In through the metal chelator, diethylenetriaminepentaacetic acid (DTPA). The radiolabeled EGF was then conjugated to a BBB delivery vector comprised of a complex of the OX26 monoclonal antibody (MAb) to the rat transferrin receptor, which was coupled to streptavidin (SA). Following intravenous injection in rats, the 125I conjugate was rapidly degraded in vivo, while the 111In conjugate was metabolically stable. The brain delivery of [111In]DTPA-EGF-PEG3400-biotin was enabled by conjugation with OX26/SA and was optimized by co-injection of unlabeled EGF to saturate EGF receptors in the liver. The specific binding of the [111In]DTPA-EGF-PEG3400-biotin conjugated to OX26/SA to the EGF receptor was confirmed in C6 rat glioma cells, which had been transfected with a gene encoding for the human EGF receptor under the regulation of a dexamethasone-inducible promoter. In vivo studies of C6-EGFR experimental tumors in Fischer 344 rats demonstrated successful brain imaging only when the peptide radiopharmaceutical was conjugated to the BBB delivery system, although the C6-EGFR tumors did not express EGFR in vivo. In conclusion, these studies describe the molecular formulation of a peptide radiopharmaceutical that can be used for imaging brain tumors behind the BBB.     

A synthetic fragment of rat transforming growth factor alpha with receptor binding and antigenic properties

A fragment of rat transforming growth factor alpha (TGF alpha) comprising the third disulfide loop (residues 34-43) was selected as a potential antigenic and receptor binding region. Immunization of rabbits with a peptide conjugate resulted in antibodies which were specific for both the peptide and rat TGF alpha, but not for the homologous epidermal growth factor (EGF). The synthetic decapeptide exhibited low affinity for EGF receptors on human cells. Affinity was increased 100x to 0.2% of EGF or TGF alpha binding by blocking the peptide ends. The blocked decapeptide had no mitogenic activity but prevented the mitogenic effect of EGF and TGF alpha on fibroblasts. This decapeptide is an antagonist and contains an important receptor binding region of TGF alpha.     

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Heterodimerizing peptides, such as the de novo designed E5/K5 peptide pair, have several applications including as tags for protein purification or immobilization. Recently, we demonstrated that E5-tagged epidermal growth factor (EGF), when bound to a K4 expressing adenovirus, promotes retargeting of the adenovirus to EGFR expressing target cells. In this study, we present the Escherichia coli expression, refolding and purification of human EGF fused with the E5-coil (E5-coil-EGF) or with the K5-coil (K5-coil-EGF). EGF receptor phosphorylation and cell proliferation assays demonstrated that the biological activity of the coil-tagged EGF versions was comparable to that of non-tagged EGF. Additionally, analysis of the binding of E5/K5-coil-EGF to cell surface EGFR or to soluble EGFR ectodomain, as measured by cell-based binding competition assays and by SPR-based biosensor experiments, indicated that the coil-tagged EGF versions bound to EGFR with affinities similar to that of non-tagged EGF. Finally, we show that E-coil-tagged EGF, but not non-tagged EGF, can retarget a K-coil containing adenovirus to EGF receptor expressing glioblastoma tumor cells. Overall these results indicate that E. coli expression offers a practical platform for the reproducible production of fully biologically active E5/K5-coil-tagged EGF, and support applications of heterodimerizing coil-tagged ligands, e.g. the targeting of viruses or other entities such as nanoparticles to tumor cells, or growth factor immobilization on cell culture scaffolds for tissue engineering.     

Epidermal growth factor labeled beta-amanitin-poly-L-ornithine: preparation and evidence for specific cytotoxicity

Poly-L-ornithine with an average molecular weight of 32K was reacted with beta-amanitin hydroxysuccinimide ester to form an amide-linked toxin conjugate. Loading of the polymeric chain with amanitin was high, corresponding to up to 35% of the total weight. To this amatoxin vehicle we attached a targeting molecule, human recombinant leucine-21 epidermal growth factor (hrEGFL), via a disulfide-containing linker moiety. A typical average stoichiometry of the hrEGFL labeled toxin conjugate was (L-Orn)164(beta-amanitin)19(COC2H4SSC2H4CO-hrEGFL)2. The affinity for EGF receptors of hrEGFL bound in this conjugate was tested by using A 431 cells. The affinity was eight times lower than that of unsubstituted hrEGFL but regarded as high enough for studying specific toxicity effects with cells bearing EGF receptors. We found that beta-amanitin in the labeled conjugate was able to inhibit the growth of A 431 cells at a concentration of 28 nM, 80 times lower than for native beta-amanitin and 20 times lower than for poly-L-ornithine-bound beta-amanitin without the hrEGFL label. The approximately 20-fold enhancement of cytotoxicity suggests a specific internalization of the toxin conjugate mediated by the hormone label. This idea is supported by the fact that also in another transformed fibroblast cell line, with an increased though smaller number of EGF receptors than A 431 cells, the corresponding enhancement of cytotoxicity was demonstrable but less pronounced (7-fold). The hormone-mediated increase in cytotoxicity of EGF labeled poly-L-ornithine-beta-amanitin conjugates, combined with their moderate toxicity in the mouse, encourages further examination of such compounds in tumor model systems in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431

We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.     

Protein–protein interactions between SWCNT/chitosan/EGF and EGF receptor: a model of drug delivery system

Epidermal growth factor (EGF) was used as the targeting ligand to enhance the specificity of a cancer drug delivery system (DDS) via its specific interaction with the EGF receptor (EGFR) that is overexpressed on the surface of some cancer cells. To investigate the intermolecular interaction and binding affinity between the EGF-conjugated DDS and the EGFR, 50 ns molecular dynamics simulations were performed on the complex of tethered EGFR and EGF linked to single-wall carbon nanotube (SWCNT) through a biopolymer chitosan wrapping the tube outer surface (EGFR·EGF-CS-SWCNT-Drug complex), and compared to the EGFR·EGF complex and free EGFR. The binding pattern of the EGF-CS-SWCNT-Drug complex to the EGFR was broadly comparable to that for EGF, but the binding affinity of the EGF-CS-SWCNT-Drug complex was predicted to be somewhat better than that for EGF alone. Additionally, the chitosan chain could prevent undesired interactions of SWCNT at the binding pocket region. Therefore, EGF connected to SWCNT via a chitosan linker is a seemingly good formulation for developing a smart DDS served as part of an alternative cancer therapy.     

Competition of a growth stimulating-/cholecystokinin (CCK) releasing-peptide (monitor peptide) with epidermal growth factor for binding to 3T3 fibroblasts

The growth stimulating-/cholecystokinin (CCK) releasing-peptide (monitor peptide) is a peptide purified from rat bile-pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. Its multiple functions and peptide sequence suggested that it is distinct from epidermal growth factor (EGF). However, we found that the peptide competes with [125I]-EGF in the binding to Swiss 3T3 fibroblast cells to almost the same extent as unlabeled EGF does. [125I]-EGF binding was inhibited by 50% by the peptide at 82.8 ng/ml and by unlabeled EGF at 71.4 ng/ml. This suggests that the growth stimulating effect of the peptide on 3T3 fibroblasts is mediated via the EGF receptor, and also suggests that the partial homologous sequence between monitor peptide and EGF is required for the receptor binding, or that the EGF receptor has a broad ligand specificity.     

Transmembrane delivery of polypeptide growth factors bypassing the intrinsic cell surface receptors: synthesis and biological activity of a conjugate of epidermal growth factor with alpha 2-macroglobulin

Epidermal growth factor (EGF) was derivatized at the amino terminus with N-succinimidyl 3-(2-pyridyldithio)propionate and then cross-linked to the cysteinyl residues of alpha 2-macroglobulin (alpha 2M) via disulfide bonds. The EGF-alpha 2M conjugate delivered EGF into dense lysosomal fractions through binding to alpha 2M receptors in a variant of mouse Swiss/3T3 fibroblasts, NR-6, which are deficient in EGF receptors. The conjugate stimulated DNA synthesis in Swiss/3T3 cells, but it did not stimulate DNA synthesis in NR-6 cells. This differential stimulation was due to the conjugate's binding to EGF receptors since bacitracin, which completely inhibits [125I]alpha 2M binding to its receptors, inhibited conjugate binding by approximately 80%. Thus, EGF bound to and internalized through alpha 2M receptors does not function as a mediator for DNA stimulation. The mechanisms of action of the conjugate are discussed in relation to the role of receptor-mediated endocytic pathways.     

Reduced immunogenicity of beta-lactoglobulin by conjugating with chitosan

Bovine beta-lactoglobulin (beta-LG) was conjugated with chitosan (CHS) by means of a water-soluble carbodiimide to reduce the immunogenicity of beta-LG. Each beta-LG-CHS conjugate was purified by ion-exchange chromatography and hydrophobic chromatography. The conjugation between beta-LG and CHS was confirmed by SDS-PAGE, the isoelectric point of the conjugate being higher than that of beta-LG. Two types of the beta-LG-CHS conjugate were obtained with molar ratios of beta-LG to CHS of 1:1 (F1) and 1:2 (F2). Structural analyses by fluorescence measurement, ELISA with monoclonal antibodies and retinol-binding activity indicated that the conjugates had almost maintained the native structure of beta-LG. The antigenicity of the beta-LG-CHS conjugates was similar to that of beta-LG in C3H/He mice. Reduction of the immunogenicity of beta-LG was achieved by conjugation with CHS. In particular, F2 showed very low immunogenicity. B cell epitopes of beta-LG and the conjugates recognized in C3H/He mice were determined with 15-mer multi-pin peptide; the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, while the antibody response to each epitope was dramatically reduced. Conjugation of beta-LG with chitosan was effective for reducing the immunogenicity of beta-LG.     

Killing of cultured hepatocytes by conjugates of asialofetuin and EGF linked to the A chains of ricin or diphtheria toxin

A disulfide-linked conjugate between asialofetuin (ASF) and the toxic A chain (RTA) of ricin is as potent a toxin for cultured rat hepatocytes as our previously described conjugate between ASF and fragment A of diphtheria toxin (DTA). An RTA conjugate of epidermal growth factor (EGF) was a potent toxin for 3T3 cells. In contrast, EGF-DTA was essentially nontoxic for 3T3 cells. We have now examined the toxicity of EGF-RTA and EGF-DTA on cultured hepatocytes. The EGF-DTA conjugate, nontoxic to 3T3 cells, is also a potent toxin for hepatocytes. We also observed a decrease with time of culture in the sensitivity of hepatocytes to the ASF and EGF conjugates. This decrease is not a result of a decrease in EGF or asialoglycoprotein receptors.     

Reversibility of the epidermal growth factor receptor self-phosphorylation reaction. Evidence for formation of a high energy phosphotyrosine bond

The reversibility of the epidermal growth factor (EGF) receptor self-phosphorylation reaction was studied using highly purified receptor from A431 human epidermoid carcinoma cells. Self-phosphorylation is inhibited by the reaction product ADP in a dose-dependent manner exhibiting an IC50 approximately 2 microM. In addition, phosphorylated EGF receptor can be rapidly dephosphorylated in the presence of ADP. The dephosphorylation reaction results in equimolar production of ATP and loss of phosphate from the receptor. The reverse reaction is dependent on time and ADP exhibiting a t1/2 of 15 s and a Km(ADP) = 0.40 +/- 0.14 microM. The dephosphorylation reaction can be effectively inhibited by an exogenous peptide substrate for the forward reaction, i.e., the src-peptide (a synthetic peptide corresponding to one of the self-phosphorylation sites in p60v-src). This suggests that the dephosphorylation reaction is intrinsic to the EGF receptor. The equilibrium constant, K, for the self-phosphorylation reaction was estimated to be 0.5-1.6 using kinetic and reactant/product concentration analyses. Assuming that the standard free energy change, delta G0, for ATP hydrolysis is -9.5 kcal/mol, an observed delta G0 for hydrolysis of the EGF receptor phosphotyrosine bond was calculated to be -9 to -10 kcal/mol. These results indicate that the EGF receptor self-phosphorylation reaction, which appears important in the regulation of EGF receptor function, is readily reversible and that the phosphotyrosine bond formed by this reaction is of relatively high energy.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Kinetic analysis of the inhibition of the epidermal growth factor receptor tyrosine kinase by Lavendustin-A and its analogue

Lavendustin-A was reported to be a potent tyrosine kinase inhibitor of the epidermal growth factor (EGF) receptor (Onoda, T., Iinuma, H., Sasaki, Y., Hamada, M., Isshibi, K., Naganawa, H., Takeuchi, T., Tatsuta, K., and Umezawa, K. (1989) J. Nat. Prod. 52, 1252-1257). Its inhibition kinetics was studied in detail using the baculovirus-expressed recombinant intracellular domain of the EGF receptor (EGFR-IC). Lavendustin-A (RG 14355) is a slow and tight binding inhibitor of the receptor tyrosine kinase. The pre-steady state kinetic analysis demonstrates that the inhibition corresponds to a two-step mechanism in which an initial enzyme-inhibitor complex (EI) is rapidly formed followed by a slow isomerization step to form a tight complex (EI*). The dissociation constant for the initial rapid forming complex is 370 nM, whereas the overall dissociation constant is estimated to be less than or equal to 1 nM. The difference between the two values is due to the tight binding nature of the inhibitor to the enzyme in EI*. The kinetic analysis using a preincubation protocol to pre-equilibrate the enzyme with the inhibitor in the presence of one substrate showed that Lavendustin-A is a hyperbolic mixed-type inhibitor with respect to both ATP and the peptide substrate, with a major effect on the binding affinities for both substrates. An analogue of Lavendustin-A (RG 14467) showed similar inhibition kinetics to that of Lavendustin-A. The results of the pre-steady state analysis are also consistent with the proposed two-step mechanism. The dissociation constant for the initial fast forming complex in this case is 3.4 microM, whereas the overall dissociation constant is estimated to be less than or equal to 30 nM. It is a partial (hyperbolic) competitive inhibitor with respect to ATP. Its inhibition is reduced to different extents by different peptide substrates, when the peptide is added to the enzyme simultaneously with the inhibitor. When studied with the least protective peptide, K1 (a peptide containing the major autophosphorylation site of the EGF receptor), RG 14467 acts as a hyperbolic noncompetitive inhibitor with respect to the peptide.     

Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery.

With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF-PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.     

Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence

The effects of varying the cationic sequence of oligotryptophan-tagged antimicrobial peptides were investigated in terms of peptide adsorption to model lipid membranes, liposome leakage induction, and antibacterial potency. Heptamers of lysine (K7) and arginine (R7) were lytic against Escherichia coli bacteria at low ionic strength. In parallel, both peptides adsorbed on to bilayers formed by E. coli phospholipids, and caused leakage in the corresponding liposomes. K7 was the more potent of the two peptides in causing liposome leakage, although the adsorption of this peptide on E. coli membranes was lower than that of R7. The bactericidal effect, liposome lysis, and membrane adsorption were all substantially reduced at physiological ionic strength. When a tryptophan pentamer tag was linked to the C-terminal end of these peptides, substantial peptide adsorption, membrane lysis, and bacterial killing were observed also at high ionic strength, and also for a peptide of lower cationic charge density (KNKGKKN-W5). Strikingly, the order of membrane lytic potential of the cationic peptides investigated was reversed when tagged. This and other aspects of peptide behavior and adsorption, in conjunction with effects on liposomes and bacteria, suggest that tagged and untagged peptides act by different lytic mechanisms, which to some extent counterbalance each other. Thus, while the untagged peptides act by generating negative curvature strain in the phospholipid membrane, the tagged peptides cause positive curvature strain. The tagged heptamer of arginine, R7W5, was the best candidate for E. coli membrane lysis at physiological salt conditions and proved to be an efficient antibacterial agent.     

Dendrimer-epidermal growth factor conjugate displays superagonist activity

Binding of ligands on to epidermal growth factor receptor (EGFR) can stimulate cell growth; therefore, any application employing EGF as a targeting ligand for a "drug carrier" must evaluate the effect of the conjugate on cell growth. We report the synthesis and in vitro biological activity of EGF molecules coupled to a fluorescein-labeled polyamidoamine dendrimer. The conjugate bound and internalized into several EGFR-expressing cell lines in a receptor-specific fashion. The conjugate effectively induced EGFR phosphorylation and acted as a superagonist by stimulating cell growth to a greater degree than free EGF. Concomitant administration of the chemotherapeutic drug methotrexate completely inhibited cell growth to a degree similar to its effect in the absence of the conjugate. Thus, dendrimer-EGF conjugates serve as EGFR superagonists, but this activity can be overcome by chemotherapeutic drugs. The agonist activity of these materials must be taken into consideration when using EGF conjugates for imaging applications.