Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S469

taining latent HIV proviruses. We found that Oxamflatin activateHIV-1 gene expression in these latently infected cells by 2-7-foldover background levels, as analyzed by flow cytometry. Chromatinimmunoprecipitation (ChIP) assays revealed that scriptaid increasethe acetylation level of H3 at the nucleosome 1(nuc-1) site of theHIV-1 long terminal repeat (LTR) compared to mock treatment. Wealso found that Oxamflatin synergize with TNF-( or prostratin toactivate the HIV-1promoter and was showed to be relatively lowtoxicity compared to TSA. These studies suggest that Oxamflatinhave potential as drug candidates to explore as potential antila-tency therapies

doi:10.1016/j.jbiotec.2010.09.697

[P-M.110]

Characterization of �-Tubulin cDNA(s) from Taxus baccata

H. Ashourion, S. Abolmaali ∗, Z. Fooladvand

Shahid Beheshti University, Islamic Republic of IranKeywords: �-Tubulin; Taxus baccata; Paclitaxel; Anticancer

Cancer is the second frequent disease and the third cause ofdying in Iran. The complicated mechanisms for cell division andalso non selective function of chemotherapy made it a disease withextremely expensive treatments. Some plant secondary metabo-lites disrupt microtubule dynamic and drive the treated cells todeath. The compounds are commonly employed as anti-cancers.Paclitaxel is a mitotic inhibitor produced by different species ofTaxus. Paclitaxel stabilizes microtubules and as a result, interfereswith the normal breakdown of microtubules during cell division.The drug is used to treat patients with lung, ovarian, breast cancer,head and neck cancer, and advanced forms of Kaposi’s sarcoma.Extremely low amount of paclitaxel in Taxus species and increaseddemand for chemotherapy are motives to scale up its productionby biotechnological approaches. The goal of this investigation isto characterize �-tubulin gene family in Taxus baccata. The datawill be utilized for genetic engineering of the plant to enhancethe amount of paclitaxel. 24 �-tubulin coding sequence of plants,mainly from Arabidopsis thaliana, were obtained from NCBI genebank. The sequences were aligned and 14 specific forward andreverse oligos designed based on the most conservative part of thesequences. Total RNA from leaves of Taxus baccata were extractedand the cDNAs synthesized using oligodT plus specific reverse oli-gos. The amplified cDNAs has been cloned in pJET cloning vector.The PCR and restriction digestion analyses have been indicatedthree different patterns. Sequencing analyses are under processing.

doi:10.1016/j.jbiotec.2010.09.698

[P-M.111]

Optimization of soluble human Interleukin-2 production inE. coli with coexpression of molecular chaperons in the coldinduced system

T. Gotoh ∗, T. Horii, K-I. Kikuchi

Akita University, JapanKeywords: cold shock; molecular chaperone; interleukin-2

The expression of eukaryotic proteins in Escherichia coli oftenyields misfolded insoluble aggregates that are known as inclusionbodies. To overcome this problem, the expression conditions wereimproved by cultivating alternate E. coli strains at low temper-ature, fusion to soluble proteins, and coexpression of molecular

chaperones, which participate in the correct folding of prematureproteins. The present study aimed to optimize the soluble expres-sion of recombinant human interleukin-2 (hIL-2) in E. coli, theprotein which increases the growth and activity of T lymphocytesand B lymphocytes and affects the development of the immune sys-tem, using the cold induced system with coexpression of molecularchaperones.

We constructed recombinant E. coli strains, which carry a coldinduced expression vector for human interleukin-2 (hIL-2) and vec-tors for coexpression of molecular chaperones (GroEL, GroES, DnaK,DnaJ, GrpE, and trigger factor). The E. coli cells were cultured inLB broth medium containing 50 �g/ml of ampicillin and 20 �g/mlof chloramphenicol at 37 ◦C. The expression of chaperones wasinduced by L-arabinose or tetracycline when OD600 reached 0.3.At 30 min later (at about OD600 of 0.5) the temperature was rapidlylowered to 15 ◦C (cold shock) to induce the expression of hIL-2,and then the culture was continued at 15 ◦C. At 24 h, the cells weredisrupted by sonication, and soluble and insoluble fractions wereanalyzed for hIL-2 by SDS-PAGE and western blotting with anti-hIL-2 antibody. A sandwich ELISA was used for the quantificationof hIL-2. As a result, the coexpression of trigger factor was foundto most effectively yield soluble hIL-2. The culture conditions forthe production of soluble hIL-2 were optimized through an assort-ment of modifications designed to vary the cold shock time for theexpression of hIL-2 and the induction time for the coexpression oftrigger factor.

doi:10.1016/j.jbiotec.2010.09.699

[P-M.112]

Stem cells and biomaterial interactions for neural tissue engi-neering models: A matter of mechanotransduction

D. D’Angelo 1,∗, I. Armentano 2, R. Tiribuzi 1, S. Mattioli 2, U. Reale 3,S. Montesano 1, L. Crispoltoni 1, G.G. Cerulli 3, J.M. Kenny 2, C.A.Palmerini 4, S. Martino 1, A. Orlacchio 1

1 Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Uni-versity of Perugia, Italy2 Materials Engineering Centre, University of Perugia, Italy3 Department of Orthopedics and Traumatology, University of Perugia,Italy4 Dipartimento di Medicina Interna, University of Perugia, Italy

Aim: this study attempted to approach the tissue engineering“hot topics” from mechanotrasduction, so as to understand what itneeds to design a “tissue engineering-model”

From the last few years we are developing stem cells regener-ative medicine applications for degenerative disorder therapy. Inthis contest we are investigating whether the chemical/physicalproperties of peculiar biomaterials could drive the stem cell dif-ferentiation. In this work we have explored the way by which thedesign of surface topography, through mechanotransduction, couldstimulate stem cell differentiation towards neural lineage.

We used the hydrogenated amorphous carbon (a-C:H) withthree different nanoscale groove topographies as mechanotrans-ducer stimuli and the brain derived neurotrophic factor (BDNF) asa biochemical inducer, to generate neural cells from human bonemarrow mesenchymal stem cells (hBM-MSCs) in vitro.

Results: The simultaneous presence of a-C:H nanogrooves andBDNF on the substrate surface resulted in the highest percentage ofneuronal differentiated cells. Morevoer, we demonstrated that onlythe surface topography with intermediate dimensions was able toinduce hBM-MSCs to acquire neuronal characteristic even in theabsence of BDNF. On the other hand the other a-C:H nanogroovesdimensions tested failed to induce stem cells to acquire neuronal

S470 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

properties validating the active role of the optimal nanostructuredimensions on the hBM-MSCs responses

Acknowledgments

This study was supported by the Italian Ministero della Salute(grant no. RF-UMB-2006-339457 to A.O.), the Italian FondazioneCassa di Risparmio di Perugia (grant no. 2009.020.0050 to A.O.),the Italian Ministero dell’Istruzione, dell’Università e della Ricerca(grants: FIRB Idea Progettuale no. RBIP06FH7J 002 and PRIN no.20084XRSBS 001 to A.O.), as well as the Istituto Nazionale Biostrut-ture e Biosistemi.

doi:10.1016/j.jbiotec.2010.09.700

[P-M.113]

A computational approach to kinome-wide binding affinityprofiling of small-molecular inhibitors using dynamic confor-mations of kinases

Q. Huang ∗, L. Yu, X. Feng, M. Xu, B. Wan, L. Yu

Fudan University, ChinaKeywords: kinase profiling; kinase inhibitor selectivity; small-molecular binding affinity; molecular modelling

Selectivity is important for small-molecular inhibitors of proteinkinases. Recently kinome-wide profiling of small-molecular bind-ing affinity has become one of main experimental approaches todiscovery of selective kinase inhibitors. To conduct the profiling ina relatively rapid and cheap way, it is very interesting to developcomputational method to accurately predict the binding affinityprofiles of inhibitors to all human kinases. Here we present a com-putational approach for accurately predicting such kinome-widebinding affinities of small molecules. To the end, structural modelsof catalytic domains of about 500 human protein kinases were firstbuilt by homology modelling and refined by high-resolution mod-elling protocols with Rosetta. Then, nanosecond-scale moleculardynamics (MD) simulations with explicit solvent representationwere performed to simulate the kinases in complex with ATP inactive sites, in order to mimic the active forms of kinases. To eval-uate the binding affinities of small molecules, for each kinase 25conformational snapshots were extracted from the MD trajectoriesto form a dynamic conformational ensemble for molecular dock-ing of small-molecular ligands. With this conformational ensemble,binding free energies of a small molecule to all 25 conforma-tions of a given kinase were calculated with the molecular dockingprogram AutoDock Vina, and the average value was used to quan-titatively describe the binding affinity of the small molecule to thegiven kinase. To test this approach, we calculated the kinome-widebinding affinity profiles for clinical kinase inhibitors. The compar-ison of calculated values with experimental data showed that ourapproach is a very promising tool to predict the kinase inhibitorselectivity.

doi:10.1016/j.jbiotec.2010.09.701

[P-M.114]

Plants and plant genes for the development of cancer vaccines

S Massa 1,2,3,∗, A Venuti 1,2,3, L Spanò 1,2,3, R Franconi 1,2,3

1 ENEA, Italian National Agency for New Technologies, Energy andSustainable Economic Development, C.R. Casaccia, Plant Genetics andGenomics Section, Italy2 Regina Elena Cancer Institute, Laboratory of Virology, Italy3 University of L’Aquila, Basic and Applied Biology Department, ItalyKeywords: Cancer Immunotherapy; plant proteins; plant expres-sion systems; launch vector

Introduction: 1) Immunotherapy against Human Papillomavirus(HPV) cancers is seeking for safe/effective vaccines. Linkingtumor-specific antigens (HPV16 E7) to molecules increasing their‘visibility’ represents a strategy to force the immune system tofight cancer. We focused on plant proteins as innovative immune-modulators with few clinical use constraints.

2) the E7 antigen can be expressed in numerous systems: it isessential to determine which system offers the most advantages forproduction. We have exploited an accelerated production platformapplicable for expressing vaccine antigens that is based on a ‘launchvector’ enabling the use of non-genetically modified plants for tar-get production and, so, creating a highly competitive platform thatbrings a new concept to biomanufacturing.

Methods: 1) Ribosome-inhibiting proteins have features(immunogenicity, inflammation and apoptosis induction, detri-mental in their former use as immunotoxins) that might beadvantageous in tumor therapy. A saporin non toxic mutant wasused as a carrier for the E7GGG gene in the context of a geneticvaccine (Italian Patent RM2009A000383).

2) We engineered the HPV16 E7 sequence as a fusion to �-1,3-1,4-glucanase (LicKM) of Clostridium thermocellum and producedit in Nicotiana benthamiana plants using this launch vector (leavesand root cultures).

Results and discussion: 1) Preliminary results show that fusionconstructs are able to induce E7-specific IgGs, CTLs and Th1cytokine-mediated inflammation affecting the growth of E7-expressing tumours in mice, demonstrating that (mutant) plantgenes hold promise to obtain humoral/cell-mediated specificimmune responses.

2) The purified target antigen induced E7-specific IgG andcytotoxic T-cell responses inhibiting tumor development follow-ing challenge with an E7-expressing tumor cell line, even ina novel orthotopic mouse model. These data demonstrate thepotential of this plant-based platform for producing human thera-peutic vaccines. Especially root bioreactor culture is the future keystep towards commercial production of bioactive therapeutics byplant biotechnology being a fast-growing, contained biotechnol-ogy.

doi:10.1016/j.jbiotec.2010.09.702

[P-M.115]

AA Sequencing, Glycosylation and Phosphorylation SiteCharacterization of Human Serum Clusterin using LiquidChromatography-High Resolution Mass Spectrometry

S. Sidoli ∗, L. Elviri, M. Careri, A. Mangia, F. Rizzi, S. Bettuzzi

Università degli Studi di Parma, Italy

Introduction: An advanced method for peptide mixture analysiswas developed and applied to characterization of human serumclusterin (sCLU), a glycoprotein secreted in all biological fluids