A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

The single EGF-like domain of mouse PAMR1 is modified by O-Glucose, O-Fucose and O-GlcNAc.

Epidermal growth factor-like domains (EGF-LDs) of membrane and secreted proteins can be modified by N-glycans and/or potentially elongated O-linked monosaccharides such as O-glucose (O-Glc) found at two positions (O-Glc 1 and O-Glc2), O-fucose (O-Fuc) and O-N-acetylglucosamine (O-GlcNAc). The presence of three O-linked sugars within the same EGF-LD, such as in EGF-LD 20 of NOTCH1, has rarely been evidenced. We searched in KEGG GENES database to list mouse and human proteins with an EGF-LD sequence including one, two, three or four potential O-glycosylation consensus sites. Among the 129 murine retrieved proteins, most had predicted O-fucosylation and/or O-GlcNAcylation sites. Around 68% of EGF-LDs were subjected to only one O-linked sugar modification and near 5% to three modifications. Among these latter, we focused on the peptidase domain-containing protein associated with muscle regeneration 1 (PAMR1), having only one EGF-LD. To test the ability of this domain to be glycosylated, a correctly folded EGF-LD was produced in Escherichia coli periplasm, purified and subjected to in vitro incubations with the recombinant O-glycosyltransferases POGLUT1, POFUT1 and EOGT, adding O-Glc1, O-Fuc and O-GlcNAc, respectively. Using click chemistry and mass spectrometry, isolated PAMR1 EGF-LD was demonstrated to be modified by the three O-linked sugars. Their presence was individually confirmed on EGF-LD of full-length mouse recombinant PAMR1, with at least some molecules modified by both O-Glc1 and O-Fuc. Overall, these results are consistent with the presence of a triple O-glycosylated EGF-LD in mouse PAMR1.

Expression, purification and functional identification of the modified hEGF protein.

Human epidermal growth factor (hEGF) plays an important role in the growth and division of epithelial cells and has good application prospects in skin-related injuries and diseases. Weak skin penetration and rapid clearance of hEGF in skin via the mononuclear phagocyte system have restricted the application of hEGF. To overcome these shortcomings, the recombinant gene TAT-hEGF-CD47 was constructed in our experiments, and the fusion protein TAT-hEGF-CD47 was expressed, purified and renatured. The cell proliferation-promoting function, skin penetration and concentration of TAT-hEGF-CD47 in skin after its application were determined. The results showed that TAT-hEGF-CD47 effectively promoted human skin fibroblast and skin epithelial cell proliferation, and the proliferation-promoting effect was positively correlated with the TAT-hEGF-CD47 concentration. After administration to the skin, TAT-hEGF-CD47 effectively penetrated the epidermal layer of the skin because of the TAT domain and stayed in the skin for a long time because the CD47 fragment slowed its clearance via the mononuclear phagocytic system. In conclusion, TAT-hEGF-CD47 exhibits high cell proliferation-promoting activity, high skin penetration efficiency and long retention time in skin and has laid the foundation for its wide application in skin repair, ulcer, diabetes and even cancer treatments.

Generation of a soluble and stable apoptin-EGF fusion protein, a targeted viral protein applicable for tumor therapy.

A promising candidate for tumor targeted toxins is the chicken anemia-derived protein apoptin that induces tumor-specific apoptosis. It was aimed to design a novel apoptin-based targeted toxin by genetic fusion of apoptin with the tumor-directed ligand epidermal growth factor (EGF) using Escherichia coli as expression host. However, apoptin is highly hydrophobic and tends to form insoluble aggregates. Therefore, three different apoptin-EGF variants were generated. The fusion protein hexa-histidine (His)-apoptin-EGF (HAE) was expressed in E. coli and purified under denaturing conditions due to inclusion bodies. The protein solubility was improved by maltose-binding protein (MBP) or glutathione S-transferase. The protein MBP-apoptin-EGFHis (MAEH) was found favorable as a targeted toxin regarding final yield (4-6 mg/L) and stability. MBP was enzymatically removed using clotting factor Xa, which resulted in low yield and poor separation. MAEH was tested on target and non-target cell lines. The targeted tumor cell line A431 showed significant toxicity with an IC50 of 69.55 nM upon incubation with MAEH while fibroblasts and target receptor-free cells remained unaffected. Here we designed a novel EGF receptor targeting drug with high yield, purity and stability.

High Efficient Expression and Purification of Human Epidermal Growth Factor in Arachis Hypogaea L.

BACKGROUND: Human epidermal growth factor (hEGF) has drawn intense research attention due to its potential ability to promote healing of serious injuries, such as cuts, burns, and diabetic ulcers. Although hEGF displays prospective clinical value, the growth factor is restricted to the treatment of chronic diabetic ulcers because of its high production cost. METHODS: Leguminous plant peanut (Arachis hypogaea L.) hairy roots contain relatively few toxic and harmful substances, and tested as an excellent production system for hEGF in our study. To explore the possibility of hEGF expression in peanut, hEGF overexpression hairy roots were obtained by infecting leaves with Agrobacterium rhizogenes R1601. RESULTS: The maximum transgenic hairy roots inducing rate was 82%. Protein purification and mass spectrometry assays showed that the protein expressed in peanut hairy roots was identified as hEGF. Furthermore, Methylthiazolyldiphenyl-tetrazolium bromide assay showed that hEGF promoted HL-7702 liver cells proliferation, which indicate that hEGF has biological activity and non-toxic on human cells. CONCLUSION: Our results demonstrate the capacity of peanut hairy root cultures as a controlled, sustainable, and scalable production system that can be induced to produce valued human proteins, such as hEGF.

Microfluidic platform for single cell analysis under dynamic spatial and temporal stimulation.

Recent research on cellular responses is shifting from static observations recorded under static stimuli to real-time monitoring in a dynamic environment. Since cells sense and interact with their surrounding microenvironment, an experimental platform where dynamically changing cellular microenvironments should be recreated in vitro. There has been a lack of microfluidic devices to support spatial and temporal stimulations in a simple and robust manner. Here, we describe a microfluidic device that generates dynamic chemical gradients and pulses in both space and time using a single device. This microfluidic device provides at least 12h of continuous stimulations that can be used to observe responses from mammalian cells. Combination of the microfluidic de-vice with live-cell imaging facilitates real-time observation of dynamic cellular response at single cell level. Using stable HEK cells with biosensors, ERK (Extracellular signal-Regulated Kinase) activities were observed un-der the pulsatile and ramping stimulations of EGF (Epidermal Growth Factor). We quantified ERK activation even at extremely low EGF concentration (0.0625µg/ml), which can not be observed using conventional techniques such as western blot. Cytoskeleton re-arrangement of the 3T3 fibroblast (stable transfection with Lifeact-GFP) was compared under abrupt and gradually changing gradient of PDGF.

Novel bis(5-methyltetrazolium)amine ligand-bonded stationary phase with reduced leakage of metal ions in immobilized metal affinity chromatography of proteins.

Immobilized metal affinity chromatography (IMAC) has been widely used for the specific separation of biopolymers. However, leakage of metal ions from IMAC adsorbents is of concern in IMAC. In this study, we designed a novel tridenate bis(5-methyltetrazolium)amine (BMTA) to reduce the leakage of metal ions by improving the affinity to immobilized metal ions. The ligand was bonded onto silica via three-step reaction to prepare a high-performance IMAC stationary phase. The chromatographic behaviors of ribonuclease A, cytochrome c, and lysozyme on the Cu(II)-, Ni(II)-, and Zn(II)-chelated stationary phase were investigated with respect to pH effect and elution with an imidazole gradient. The retention times of these three proteins increased by increasing the pH of the mobile phase but decreased by increasing the concentration of the competitive displacer. The retaining strength of the three proteins on the chelated stationary phase were in the order Cu(II) > Ni(II) > Zn(II). The behavior of these three proteins was consistent with the properties of a typical IMAC. The BMTA ligand exhibited a much stronger affinity for Cu(II) and Ni(II) than iminodiacetic acid (IDA), which is often regarded as a standard tridentate IMAC ligand. Quantum mechanical calculations at the B3LYP/6-31G level were used to image the coordination mode of the protein-metal ions-BMTA complex. In addition, a fused histidine-tagged cecropin b-human epidermal growth factor (CB-EGF) from Escherichia coli crude extract was purified by the Ni(II)-chelated stationary phase, and the purity of the CB-EGF was determined to be at least 90 %. These results suggest that the BMTA ligand may have potential applications in the preparation of therapeutics. Graphical Abstract A novel ligand of tridenate bis(5-methyltetrazolium)amine (BMTA) was designed to reduce the leakage of metal ions from the column in immobolized metal affinity chromatography (IMAC).

Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

Epidermal Growth Factor Removal or Tyrphostin AG1478 Treatment Reduces Goblet Cells & Mucus Secretion of Epithelial Cells from Asthmatic Children Using the Air-Liquid Interface Model.

RATIONALE: Epithelial remodelling in asthma is characterised by goblet cell hyperplasia and mucus hypersecretion for which no therapies exist. Differentiated bronchial air-liquid interface cultures from asthmatic children display high goblet cell numbers. Epidermal growth factor and its receptor have been implicated in goblet cell hyperplasia. OBJECTIVES: We hypothesised that EGF removal or tyrphostin AG1478 treatment of differentiating air-liquid interface cultures from asthmatic children would result in a reduction of epithelial goblet cells and mucus secretion. METHODS: In Aim 1 primary bronchial epithelial cells from non-asthmatic (n = 5) and asthmatic (n = 5) children were differentiated under EGF-positive (10 ng/ml EGF) and EGF-negative culture conditions for 28 days. In Aim 2, cultures from a further group of asthmatic children (n = 5) were grown under tyrphostin AG1478, a tyrosine kinase inhibitor, conditions. All cultures were analysed for epithelial resistance, markers of differentiation using immunocytochemistry, ELISA for MUC5AC mucin secretion and qPCR for MUC5AC mRNA. RESULTS: In cultures from asthmatic children the goblet cell number was reduced in the EGF negative group (p = 0.01). Tyrphostin AG1478 treatment of cultures from asthmatic children had significant reductions in goblet cells at 0.2 µg/ml (p = 0.03) and 2 µg/ml (p = 0.003) as well as mucus secretion at 2 µg/ml (p = 0.04). CONCLUSIONS: We have shown in this preliminary study that through EGF removal and tyrphostin AG1478 treatment the goblet cell number and mucus hypersecretion in differentiating air-liquid interface cultures from asthmatic children is significantly reduced. This further highlights the epidermal growth factor receptor as a potential therapeutic target to inhibit goblet cell hyperplasia and mucus hypersecretion in asthma.

The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineage-specific novelties.

Proteins that are occluded within the molluscan shell, the so-called shell matrix proteins (SMPs), are an assemblage of biomolecules attractive to study for several reasons. They increase the fracture resistance of the shell by several orders of magnitude, determine the polymorph of CaCO(3) deposited, and regulate crystal nucleation, growth initiation and termination. In addition, they are thought to control the shell microstructures. Understanding how these proteins have evolved is also likely to provide deep insight into events that supported the diversification and expansion of metazoan life during the Cambrian radiation 543 million years ago. Here, we present an analysis of SMPs isolated form the CaCO(3) shell of the limpet Lottia gigantea, a gastropod that constructs an aragonitic cross-lamellar shell. We identified 39 SMPs by combining proteomic analysis with genomic and transcriptomic database interrogations. Among these proteins are various low-complexity domain-containing proteins, enzymes such as peroxidases, carbonic anhydrases and chitinases, acidic calcium-binding proteins and protease inhibitors. This list is likely to contain the most abundant SMPs of the shell matrix. It reveals the presence of both highly conserved and lineage-specific biomineralizing proteins. This mosaic evolutionary pattern suggests that there may be an ancestral molluscan SMP set upon which different conchiferan lineages have elaborated to produce the diversity of shell microstructures we observe nowadays.

Extraction of PLGA-microencapsulated proteins using a two-immiscible liquid phases system containing surfactants.

PURPOSE: The extraction of proteins from PLGA/PLA microspheres by a two-immiscible liquid phases system with the addition of surfactants was investigated. METHODS: First, the extraction without surfactants and the interaction between proteins (IFN-α2b and EGF) and empty microspheres (PLGA or PLA) was studied. Next, proteins stability in presence of different surfactants was evaluated by: (1) bicinchoninic acid protein assay, (2) reversed phase-high performance liquid chromatography, and (3) enzyme-linked immunosorbent assay. Then, proteins were extracted with PBS/dichloromethane including selected surfactants and characterized by the above mentioned techniques, biological activity tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry. RESULTS: Without surfactants, protein recovery was only 27-43% for IFN-α2b and 58-73% for EGF. Protein content in solutions incubated with blank microspheres decreased to 66% for IFN-α2b and 86% for EGF. It was only possible to quantify the EGF and IFN-α2b in the same manner as in PBS alone when the surfactant added was Pluronic F-68 and SDS, respectively. Addition of these surfactants allowed the complete isolation of both biomolecules from the microspheres. The extraction procedure did not affect the encapsulated proteins. CONCLUSION: Proteins can be quantitatively extracted, without changes, from PLGA/PLA microspheres using PBS/dichloromethane system that include an appropriate surfactant.

5-Fluorocytosine combined with Fcy-hEGF fusion protein targets EGFR-expressing cancer cells.

Human epithelial cancers account for approximately 50% of all cancer deaths. This type of cancer is characterized by excessive activation and expression of the epidermal growth factor receptor (EGFR). The EGFR pathway is critical for cancer cell proliferation, survival, metastasis and angiogenesis. The EGF-EGFR signaling pathway has been validated as an important anticancer drug target. Increasing numbers of targeted therapies against this pathway have been either approved or are currently under development. Here, we adopted a prodrug system that uses 5-fluorocytosine (5-FC) and human EGF (hEGF) fused with yeast cytosine deaminase (Fcy) to target EGFR-overexpressing cancer cells and to convert 5-FC to a significantly more toxic chemotherapeutic, 5-fluorouracil (5-FU). We cloned and purified the Fcy-hEGF fusion protein from Pichia pastoris yeast. This fusion protein specifically binds to EGFR with a similar affinity as hEGF, approximately 10 nM. Fcy-hEGF binds tightly to A431 and MDA-MB-468 cells, which overexpress EGFR, but it binds with a lower affinity to MDA-MB-231 and MCF-7, which express lower levels of EGFR. Similarly, the viability of EGFR-expressing cells was suppressed by Fcy-hEGF in the presence of increasing concentrations of 5-FC, and the IC(50) values for A431 and MDA-MB-468 were approximately 10-fold lower than those of MDA-MB-231 and MCF-7. This novel prodrug system, Fcy-hEGF/5-FC, might represent a promising addition to the available class of inhibitors that specifically target EGFR-expressing cancers.

A novel virally inactivated human platelet lysate preparation rich in TGF-beta, EGF and IGF, and depleted of PDGF and VEGF.

There is emerging interest in the use of standardized virally inactivated human platelet lysate preparations rich in GFs (growth factors) for cell cultures, cell therapy and clinical applications. In the present paper, we report a simple process to prepare a virally inactivated platelet lysate preparation rich in TGF-beta1 (transforming growth factor-beta1), EGF (epidermal growth factor) and IGF (insulin-like growth factor) and depleted of PDGF (platelet-derived growth factor) and VEGF (vascular endothelial growth factor). Apheresis platelet concentrates were treated by the S/D (solvent/detergent) viral inactivation procedure, then subjected to an oil extraction followed by adsorption with activated charcoal and finally sterile-filtered. The resulting preparation contained a mean of 368.4, 2.4 and 54.7 ng/ml of TGF-beta1, EGF and IGF respectively. PDGF-AB and VEGF were essentially completely removed by the charcoal treatment. The mean albumin, IgG, IgM and IgA and fibrinogen contents were approx. 40.0, 8.5, 0.87, 1.66 and 2.65 mg/ml respectively, cholesterol and triglycerides were at 15 and 20.7 mg/ml respectively and TnBP (tri-n-butyl phosphate) and Triton X-45 were at 8.7 and 8.8 p.p.m. respectively. Supplementing MEM (minimum essential medium) with 1-10% of this S/D-treated platelet lysate promoted the proliferation of MG63 and SIRC cell lines as well as, or better than, 10% (v/v) FBS (fetal bovine serum), as based on the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. The process used to prepare such S/D-treated platelet lysates is easily scalable for industrial production. Our results open up the possibility to evaluate the potential of this new preparation for stem cell expansion and/or bone tissue engineering and regeneration.

Batch and fed-batch cultivation for excretive production of human epidermal growth factor (hEGF) with recombinant E. Coli K12 system.

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32 degrees C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.

Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli.

Amongst the various endogenous growth factors, epidermal growth factor (EGF) plays an important role in normal wound healing of tissue such as skin, cornea and gastrointestinal tract. Various studies have proved that supplementing recombinant human EGF (rhEGF) results in significant augmentation of wound healing. In the present work, a high level expression system with poly-arginine sequences was used for the production of recombinant human EGF (rhEGF) as inclusion bodies. The inclusion bodies were solubilized and the protein was refolded by using expanded-bed adsorption chromatography. The renatured protein was digested with appropriate concentration of trypsin and subsequently the digested rhEGF is purified by passing through ion-exchange chromatography (Toyopearl-SP) to obtain a biologically active protein. This process is the shortest process with reduced number of steps of purification, eliminates the usage of preparative reversed phase HPLC (RP-HPLC) for final purification, which is an expensive technique. The purified protein was analyzed by RP-HPLC, showing a purity > 99% and size exclusion chromatography profile shows that there are minimal aggregates, with 99% renatured active protein. The purified rhEGF showed a specific activity of 5 x 10(5) IU/mg protein, in comparison with NIBSC standard (1st International Standard of rDNA-derived EGF, Code 91/530). The process has been successfully adopted at 100 L fermentation scale and the rhEGF based formulation has been commercialized with brand name REGEN D, with excellent clinical results.

Cell surface restriction of EGFR by a tenascin cytotactin-encoded EGF-like repeat is preferential for motility-related signaling.

The 14th EGFL-repeat (Ten14) of human tenascin cytotactin activates the epidermal growth factor receptor (EGFR) with micromolar affinity; however, unlike EGF, Ten14-mediated activation of EGFR does not lead to receptor internalization. As the divergent signaling pathways downstream of EGFR have been shown to be triggered from plasma membrane and cytosolic locales, we investigated whether Ten14-mediated surface restriction of EGFR resulted in altered biochemical and cellular responses as compared to EGF. Molecules associated with migratory cascades were activated to a relatively greater extent in response to Ten14, with very weak activation of proliferation-associated cascades. Activation of phospholipase C gamma (PLCgamma) and m-calpain, associated with lamellipod protrusion and tail retraction, respectively, were noted at even at sub-saturating doses of Ten14. However, activation of ERK/MAPK, p90RSK, and Elk1, factors affecting proliferation, remained low even at high Ten14 concentrations. Similar activation profiles were observed for EGF-treated cells at 4 degrees C, a maneuver that limits receptor internalization. We demonstrate a concurrent effect of such altered signaling on biophysical responses-sustained migration was observed at levels of Ten14 that activated PLCgamma, but did not stimulate proliferation significantly. Here, we present a novel class of EGFR ligands that can potentially signal as a part of the extracellular matrix, triggering specific intracellular signaling cascades leading to a directed cellular response from an otherwise pleiotropic receptor. This work extends the signaling paradigm of EGFL repeat being presented in a restricted fashion as part of the extracellular matrix.

Chimeric toxins inhibit growth of primary oral squamous cell carcinoma cells.

Treatment of oral squamous cell carcinoma (OSCC) is currently based on surgery and radiotherapy. Prolongation of the survival time of patients with progressing tumors is infrequently achieved. To improve the therapeutic options, targeted therapies are a favorable alternative. Therefore, we analyzed the effect of a chimeric toxin (CT) named SE consisting of the epidermal growth factor and the plant protein toxin saporin from Saponaria officinalis. A second construct (SA2E) additionally contains a peptidic adapter designed to enhance efficacy of the CT in vivo and to reduce side effects. The IC(50) values for an OSCC cell line (BHY) were 0.27 nM and 0.73 nM for SE and SA2E, respectively, while fibroblasts remained unaffected. To investigate primary tumor cells, we developed a technique to analyze freshly prepared OSCC cells of 28 patients in a stem cell assay directly after surgery. Cells were treated for 1 h with the CTs, subsequently seeded into soft agar and colony growth determined after 1-2 weeks In spite of the short time of CT incubation, the amount of colonies was reduced to about 78% by 10 nM and to 69% by 100 nM of either toxin. A combined application of 10 nM SA2E with a saponin from Gypsophila paniculata reduced the amount of surviving cells to 68%. The results demonstrate the impact of the CTs on OSCC cells and depict that the stem cell assay is suitable to determine the potential of anti-tumor drugs before studies in vivo will be initiated.

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography.

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.

Selection of internalizing ligand-display phage using rolling circle amplification for phage recovery.

Selection of phage libraries against complex living targets such as whole cells or organs can yield valuable targeting ligands without prior knowledge of the targeted receptor. Our previous studies have shown that noninfective multivalent ligand display phagemids internalize into mammalian cells more efficiently than their monovalent counterparts suggesting that cell-based selection of internalizing ligands might be improved using multivalently displayed peptides, antibodies or cDNAs. However, alternative methods of phage recovery are needed to select phage from noninfective libraries. To this end, we reasoned that rolling circle amplification (RCA) of phage DNA could be used to recover noninfective phage. In feasibility studies, we obtained up to 1.5 million-fold enrichment of internalizing EGF-targeted phage using RCA. When RCA was applied to a large random peptide library, eight distinct human prostate carcinoma cell-internalizing peptides were isolated within three selection rounds. These data establish RCA as an alternative to infection for phage recovery that can be used to identify peptides from noninfective phage display libraries or infective libraries under conditions where there is the potential for loss of phage infectivity.