Oral Presentation Abstract Information (posters below)

Thermostability of xylose (glucose) isomerase and its importance in the production of high fructose corn syrup
Kevin L. Epting1, Claire Vieille2, Rockey K. Bandlish 1, J. Gregory Zeikus 2, and Robert M. Kelly 1
1: North Caroline State University, 2:Michigan State University

Xylose isomerase (XI) catalyzes the conversion of xylose to xylulose. Its industrial importance is due to its ability to convert glucose to fructose in the manufacture of high fructose corn syrup (HFCS), hence it is also referred to as glucose isomerase. Current production of HFCS uses immobilized XIs at around 60C. This limits the conversion of glucose to fructose to 42 percent, and requires an expensive enrichment step to increase the mixture to 55 percent fructose. A thermostable XI would allow the process to be operated at higher conversion. Based on sequence analysis, XI’s have been divided into two classes, I and II. Enzymes from class I lack a fifty amino acid residue at the N-terminus that is present in those from class II. Currently, only class I enzymes are used in HFCS production. The class II enzymes include XIs from mesophiles, thermophiles, and hyperthermophiles. The sequences for the XIs are highly conserved, and due to their industrial importance much sequence and biochemical data are available, which makes XIs a good model system to study thermostability. XIs from E. coli, T. thermosulfurogenes, and T. neapolitana were examined with respect to their thermostability. During the study, it was noted that there was a noticeable reduction in the asparagine (Asp) and glutamine (Gln) composition of the thermostable XIs. Overall, hyperthermophilic proteins contain 55% fewer Gln and 28% fewer Asn then mesophilic protiens. Since both Asp and Gln are easily deaminated at higher temperature, it is not surprising that these would be reduced in thermostable enzymes. This led to the question of whether other XIs with low Asp and Gln could be more thermostable. The Bacillus licheniformis XI (BLXI) was selected for further study based on this criterion. The recombinant enzyme cloned and expressed in E. coli exhibited similar kinetic parameters as other class II XIs. However, differential scanning calorimitry (DSC) results showed the Tmelt for the active enzyme was 74C, which is significantly higher than one would expect for an organism with a growth temperature around 37C. Discussed here will be the factors that appear to contribute to the stability of XIs based on comparisons among class II versions of the enzyme with different levels of thermophilicity.

Characterization and Optimization of Mammalian G-Protein Coupled Receptors Expression in the yeast S. cerevisiae
James Butz, Anne Robinson
University of Delaware

G-protein coupled receptors (GPCRs) constitute a large class of proteins that span the plasma membrane seven times and may possess covalent modifications, such as glycosylation and disulfide bonds. A GPCR's physiological role is to recognize specific extracellular stimuli and to allow the cell to respond appropriately. Since it is believed that 60% of all medications interact with members of this family, GPCRs represent important drug targets. However, at this point in time no optimal expression system exists to help facilitate the development of new medications intended for specific receptors. Our research has focused on characterizing and optimizing the expression of two pharmacologically distinct mammalian GPCRs in S. cerevisiae, the mouse Substance P receptor and the human A2a receptor. During the characterization phase, both the fate and the activity of these two receptors were analyzed. Unlike the expression of mammalian GPCRs in E. coli, which results in the formation of insoluble aggregates, both receptors are expressed as integral membrane proteins in yeast. Furthermore, we have shown that the human A2a receptor is active and successfully trafficked to the plasma membrane. In order to produce higher titers of receptor, intracellular proteins that help fold nascent proteins (chaperones) or capable of catalyzing covalent modifications were co-expressed with the mammalian GPCRs. Since most GPCRs are glycoproteins with intramolecular disulfide bonds, a chaperone believed to interact with glycoproteins and isomerases capable of forming and rearranging disulfide bonds were chosen. Our data not only shows that the co-expression of certain chaperones or foldases can result in a two fold increase in GPCR expression, but gives us greater insight into membrane protein biogenesis.

The Effects of Dilution Rate on the Refolding of Prochymosin from Inclusion Bodies Formed in Escherichia coli
Jon Shanter, Dr. Alfred Carlson
The Pennsylvania State University

Prochymosin was refolded at various concentrations to determine its effect on the kinetics and yield of refolding. The fraction of prochymosin refolded was highly concentration dependent. As the prochymosin concentration in the renaturation solution was varied from 0.01 mg/mL to 1 mg/mL, the percent refolded decreased from 8% to less than 1%. The yield was further increased to over 15% with the use of a slow addition technique to mimic a lower protein concentration in the refolding solution. At higher concentrations such as 0.2 gm/mL a more drastic difference was seen with yields increasing as much as nine-fold. The proposed kinetic model suggests that the refolding pathway is first order passing through an intermediate, while it competes with a second order reaction that leads to reaggregated prochymosin. Escherichia coli cell DH5a was transfected with the plasmid pWHA43, and this organism was used to produce bovine prochymosin in the form of insoluble inclusion bodies. The cells were lysed and the inclusion bodies were collected in a pellet fraction. The inclusion bodies were then solubilized in an 8 M urea solution at pH 10.5. The resulting solution, containing unfolded prochymosin, was diluted ten-fold reducing the urea concentration to 0.8 M, triggering the refolding process. Reduced and oxidized glutathione were used in the refolding solution to facilitate disulfide rearrangement allowing the proteins to refold properly. The refolded prochymosin was then activated to pseudochymosin by acidification, and a milk-clotting activity assay was used to determine the concentration of activated pseudochymosin that was properly refolded.
This work was funded by the Center for Bioprocessing Research.

Ex vivo Monitoring of Baculovirus-infected Larvae Via an Optical Probe
Shannon F. Kramer, Govind Rao, Yordan Kostov, William E. Bentley
University of Maryland-College Park

Trichoplusia ni (T. ni) larvae were used as a host organism for recombinant baculoviruses expressing UV optimized green fluorescent protein (GFPuv) or GFPuv with either human interleukin-2 (hIL-2) or chloramphenicol acetyl transferase (CAT). A GFP optical probe was used to both excite the green fluorescent protein (373 nm), and subsequently monitor fluorescence emission (510 nm) in viable larvae. The amount of GFPuv present in the infected larvae was quantified using a voltmeter connected to the optical probe's photodetector. The voltage readings were significantly higher for infected versus uninfected larvae and showed a strong correlation with the amount of GFPuv being produced which in turn indicated the amount of hIL-2 or CAT present in the larvae. Furthermore, the readings obtained with the probe had less variation and required less labor than the readings obtained by fluorescent spectroscopy or Western blot analysis. Importantly, the results were obtained in real time. Therefore, the GFP optical probe, in conjunction with recombinant baculovirus expressing GFP, can be used to monitor the progress of infection in T. ni larvae and to predict the optimal harvest time for increased recombinant protein yields.

Cloning and Expression of Drosophila melanogaster beta-N-Acetyglucosaminidase Genes in S2 Cells.
Badarulhisam Abdul-Rahman1, Karen Palter2, Eric Ailor3, Julian Jones3, Nobora Tomiya1, Yuan C. Lee1 and Michael HJ. Betenbaugh3 .
1Department of Biologyk, 3Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland. 2Department of Biology, Temple University, Philadelphia, Pennsylvania.

The use of insect cells as host is a popular alternative for the expression of recombinant glycoproteins. One advantage over prokaryote or lower eucaryote system is the ability to perform protein glycosylation. However, unlike higher eucaryote such as mammalian or human, the glycoprotein produced in insect system rarely produces the complex type N-glucan. The presence of large quantity of naturally occurring non-lysosomal beta-N-acetylglucosaminidase in insect cells is believed to be one of the major reasons. This degrading enzyme specifically removed the GlcNAc on the alpha-1,3 Mannose branch and thus blocks further elongation of the N-glycan. Insect cells lacking this enzyme would be an ideal host for the production of therapeutic glycoprotien. This goal can be achieved by the use of specific inhibitor. In order to identify hexosaminidase genes in Drosophila melanogaster, a BLAST search was performed using both the Bombyx mori and Manduca Sexta sequences. Two different hexosaminidase genes were identified, DmHexol and DmHexo2, which have approximately 50% and 35% amino acid identity with the lepidopteran sequences respectively. The stably transformed S2 cells produced a significant amount of beta-N-acetylglucosaminidase enzyme, which is much higher than the basal level. Large scale expression, purification and further enzyme characterization works will also be described.

Preparation, Stability, and in vitro Performance of Vesicles made with Diblock Copolymers
James C-M. Lee, Harry Bermudez, Bohdana M. Discher, Maureen A. Sheehan,You-Yeon Won, Frank S. Bates, Dennis E. Discher
School of Engineering and Applied Science, University of Pennsylvania

Vesicles made completely from diblock copolymers - polymersomes - can be stably prepared by a wide range of techniques common to liposomes. Processes such as film rehydration, sonication, and extrusion can generate many-micron giants as well as monodisperse, ~100 nm vesicles of PEO-PEE (polyethyleneoxide - polyethylethylene). These thick-walled vesicles of polymer can encapsulate macromolecules just as liposomes can, but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal transitions and a sub-population even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an extended time with an excess of polymersomes. The effects are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG-driven micellization. The results potentiate a broad new class of technologically useful, polymer-based vesicles.

A Unified Modeling Framework for Incremental Truncation and DNA Shuffling
Gregory L. Moore, Costas D. Maranas, Stefan Lutz, Stephen J. Benkovic
The Pennsylvania State University

Combinatorial DNA libraries are widely employed to search for novel proteins for many different applications ranging from industrial enzymes to vaccines. Directed evolution experiments exploit high-throughput screening of proteins encoded by the combinatorial library to rapidly select enhanced proteins. The objective of this work is to provide quantitative guidelines for setting up the generation of combinatorial DNA libraries through DNA shuffling and incremental truncation. DNA shuffling starts with the random fragmentation of a small library of DNA sequences from different species that encode for a given protein. The fragments are reassembled through fragment annealing and polymerase extension, introducing crossovers when fragments from two different parent sequences anneal and extend. Since this annealing requires sequence identity, crossovers only occur along matching regions, leading to an uneven sampling of sequence space and perhaps omission of promising sequences. In contrast, incremental truncation for the creation of hybrid enzymes is sequence independent and creates combinatorial libraries of all possible single crossovers between two sequences as recently verified by Ostermeier et al. (1999). Although incremental truncation overcomes identity limitations, it is restricted because only a single crossover is generated per sequence pair. This implies that DNA shuffling and incremental truncation are complementary in nature. Here we propose a quantitative method for analyzing combinatorial DNA library generation that models the results of both DNA shuffling and incremental truncation. The analysis of these two protocols shows that together they are capable of producing novel multiple-crossover sequences containing crossovers that are not based on regions of sequence identity.

Green Fluorescent Protein as a Fusion Partner in S. cerevisiae: studies of the fusion effect, protein expression and localization.
Jincai Li, W.E. Bentley and Govind Rao
University of Maryland Baltimore County

Green Fluorescent Protein (GFP) has found tremendous applications in various fields in the past decade. Our lab has previously demonstrated that GFP could be used as a quantitative reporter both in E. coli and in S. cerevisiae . In this study, we further examine the use of GFP for monitoring fusion protein, i.e. Hexokinase(HXK)—GFP, expression and localization events. A multiple fusion HXK-EK-GFP-6xHis was constructed where the Histidine tag (6xHis) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that the Hexokinase-GFP fusion proteins retain their structure/enzyme activity, and most importantly, there is a linear correlation between Hexokinase activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of Hexokinase and thus did not affect the enzymatic activity of HXK. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that allowed for real-time and live observation of the fusion proteins.


POSTERS

High-Performance Chromatofocusing Using Micropellicular Column Packings
Xuezhen Kang and Douglas D. Frey
University of Maryland at Baltimore County

Micropellicular (i.e., nonporous) column packings are used in chromatofocusing to demonstrate the resolution and speed achieved when separating proteins under these conditions. Linear or concave pH gradients are produced with simple mixtures of buffering species instead of the more commonly used polyampholyte buffers. Computer- aided design methods are demonstrated for selecting the composition of the elution buffer to produce a pH gradient of a desired shape. The method is applied to the high- resolution separation of horse myoglobin and human hemoglobin variants. A useful selection of buffering species is described capable of producing pH gradients of a variety of shapes in the range between pH 9.5 and 5.5.

Design of Synthetic Gene Transfer Agents
Veena Pata, Nily Dan
Drexel University

Gene therapy is one of the most promising methods of treating genetic diseases. It involves delivery of healthy gene into the cells of the affected organ or tissues. The methods used for gene delivery can be broadly classified into two categories - viral and synthetic methods. There are certain disadvantages in using viral methods, such as immunogenic reactions, limited size of genetic inserts and non-specificity. Therefore, a lot of attention has been drawn towards non-viral or synthetic carriers. This is because, synthetic vectors offer greater ease of use, safety and there are no theoretical limitations on the amount of gene that can be packaged. However, the efficiency of gene delivery by non-viral methods is low. Hence, more research is needed to increase the efficiency of these methods. There is a strong relationship between carrier structure and its performance. Hence, effective carriers can be designed by controlling their structure. This study investigates the properties of polymer-based carriers. The carrier used is a cationic polyelectrolyte (polylysine), encapsulating a rod-like DNA agent. Monte Carlo simulations are used to determine the net charge of the complex and the number of bonds formed per DNA monomer, which determines the strength of DNA-polymer binding.

Mixed Signals: Fungal elicitor and methyl jasmonate effects on sesquiterpene cyclase promoter activation in Hyoscyamus muticus hairy roots
Colleen Merritt and Wayne R. Curtis
The Pennsylvania State University

Sesquiterpene cyclase is a key branchpoint enzyme in the isoprenoid biosynthetic pathway and catalyzes the conversion of FPP to sesquiterpenes. The sesquiterpene cyclase promoter is responsive to fungal elicitation and plant defense signal molecules such as jasmonic acid. The H. muticus sesquiterpene cyclase promoter (Hmp) was fused to the green fluorescent protein (gfp) gene, cloned into a binary vector, and used to develop H. muticus hairy roots containing the Hmp-GFP construct via Agrobacterium transformation. The objectives of this research are to determine the effects of controlled application of fungal elicitor and jasmonic acid upon localization of promoter activation in hairy roots. Specifically, we aim to observe when and where the promoter is turned on in a plant root in response to controlled elicitor application by tracing GFP fluorescence. The eliciting substance is immobilized in a slab of nutrient agar that is applied to the root tip while the remainder of the root is contacted with only nutrient agar. GFP fluorescence is recorded over 24 hours along the axis of the root. Preliminary results indicate that fungal elicitor stimulates promoter activation (GFP fluorescence) only in the region directly contacted with the elicitor. However, jasmonic acid application results in very intense fluorescence that increases with time polarly along the root axis from the contacted region toward the root tip only. We are currently investigating the transport of jasmonic acid as a secondary signal in the elicitation process. Combined with jasmonate biosynthesis inhibitor studies, this work may allude to the role of jasmonic acid in plant root defense signal transduction at long distances from the stimuli.

Tapered Fiber Optic Sensors for Measuring Cellular Physiologic Response
Hong Sang, P. Mohana Shankar, Steve Wrenn and Raj Mutharasan
Drexel University

Our long-term objective is to develop a methodology and a device to map metabolic activities of tissues and cells in a local region. Optical fibers provide a convenient and efficient means of sensing of metabolic activity in situ. In this preliminary work, we report on the results of three experiments. Single mode optic fiber, with a tapered geometry in the light path coupled to a light source, was successfully used for sensing fluorescent molecules (thioflavin S, Alexa fluor, and BCECF). Life time changes due to NAD(P)H binding with a dehydrogenase was determined. Finally, dynamics of intracellular NADH and its binding are determined using life time measurement due to changes in oxygen availability.

Characterization of Intracellular Proteolytic Activity in Pichia pastoris
Nekeisha S. Sweeney and Tracey R. Pulliam Holoman University of Maryland
Chemical and Nuclear Engineering Building
College Park, Maryland 20742-2111

Pichia pastoris is a methylotrophic yeast that has a strong, inducible promoter which can be used for protein production. It is capable of generating post-translational modifications that are more similar to eukaryotic protein modifications such as glycosylation. P. pastoris does not secrete high amounts of endogenous protein; therefore the heterologous protein secreted into the culture is relatively pure and purification is easier to accomplish. In this study we investigated the intracellular proteolytic activity during the growth and protein production stages of batch fermentations of the P. pastoris in both rich media (Yeast-extract Peptone Dextrose) and BMGY (Buffered Medium containing Glycerol) under oxygen -rich and oxygen-limited conditions. Proteolytic activity was analyzed using SDS G-PAGE. Additionally, pulsed vs. continuous methanol feed was investigated.

Measuring Cell Traction Forces on Collagen Gels
Aron Parekh and Darrell Velegol
The Pennsylvania State University

New tissue can be developed by growing cells in a polymer matrix that can later be implanted to replace defective tissue within the body. In order to understand the design of novel types of tissue, one must understand the interactions between cells and the polymer matrix in which they are growing. For example, fibroblasts, a common cell type found in connective tissue, are known to remodel collagen, a biological polymer, by exerting traction forces on the collagen to change its overall shape. These shape changes or displacements, combined with the mechanical properties of the collagen, can be used to calculate the cell traction forces. The remodeling resulting from these forces could produce materials with specific local mechanical properties ideal for tissue equivalents. In order to determine the cell traction forces from the collagen fibril displacements, the mechanical properties of collagen must be measured. Early in remodeling, the collagen is assumed to be an isotropic, elastic material in which none of its properties vary with respect to direction. A linear relationship between the displacement of the collagen fibrils and the cell traction forces can be deduced from elasticity theory. This relationship can then be utilized to calculate the cell traction forces from experimental values for the collagen fibril displacements measured by video microscopy and calculated by deformation and fitting algorithms. Preliminary results show traction forces of 5 to 25 nN in a 40 minute period.

A Fluorescence Energy Transfer Study Of Lecithin-Cholesterol Vesicles In The Presence Of Sodium Tauroursodeoxycholate
Manasa V.Gudheti, Steven P.Wrenn
Drexel University

Fluorescence energy transfer from dehydroergosterol to dansylated lecithin was used to characterize lecithin-cholesterol vesicles in the presence of the bile salt, sodium tauroursodeoxycholate. The approach is to demonstrate agreement between the fluorescence technique and conventional studies with respect to the vesicle-to- micelle transition as a function of bile salt loading. The changes in the fluorescence ratio correlated well with the conventional light scattering measurements that quantify partial micellization of vesicles as a function of bile salt loading. In addition, fluorescence energy transfer from dehydroergosterol to dansylated lecithin revealed cholesterol enrichment of vesicles and re-vesiculation of micelles at bile salt loadings for which vesicles and micelles co-existed

Anaerobic Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Enrichment Cultures Under Methanogenic Conditions
Wook Chang, Youngsoon Um and Tracey R. Pulliam Holoman
University of Maryland, College Park, Maryland 20742-2111

The widespread contamination of aquatic sediments by polycyclic aromatic hydrocarbons (PAHs) has led to the need for efficient, cost-effective anaerobic bioremediation processes. Recently, several studies demonstrated PAH degradation under nitrate- and sulfate-reducing conditions; however, very limited information is available on degradation of PAHs under methanogenic conditions. This research focuses on the characterization of PAH-degrading microbes and population dynamics for mixtures of PAHs under methanogenic conditions. Baltimore Harbor sediments were utilized to initiate anaerobic enrichment cultures in estuarine medium with naphthalene (NAP), phenanthrene (PHE), and pyrene (PYR), respectively. Both NAP and PHE degradation were detected, but not PYR. Subsequently, the NAP and PHE enrichment cultures were transferred to fresh medium and refed NAP and PHE, respectively. Again, the cultures continued to exhibit NAP and PHE degradation. The population of the PAH-degrading consortia was monitored via comparative sequence analysis of 16S rDNA. By utilizing these techniques, shifts in the microbial community as a result of further enrichment and degradation were monitored. Initial characterization of the NAP- and PHE-degrading consortium revealed 41 and 28 distinct RFLP types with universal primers and 13 and 15 with archaeal primers, respectively. Further, the predominant RFLP types exhibited the high sequence similarity to anaerobes which have been implicated in the degradation of other contaminants, such as PCB, trichlorobenzene, benzene, and hydrocarbons. This preliminary information is utilized to monitor population dynamics and biodegradation kinetics for PAH mixtures (NAP, PHE, and PYR).

Green Fluorescent Protein as a real-time indicator to monitor gene expression in bioprocess
Canghai Lu, C.Renee Albano, William E. Bentley, Govind Rao
University of Maryland Baltimore County

The use of green fluorescent protein as a reporter gene has made a broad impact in several areas especially in studies of protein trafficking, localization and expression analysis. Its many advantages are that it is small, autocatalytic, and does not require fixation, sacrifice, or the addition of any co-factors or substrates. Two characteristics of GFP, extreme stability and lag time, pose a hindrance to the application of GFP as a real time gene expression reporter in bioprocess applications. In this report, we present analytical methods which overcome these problems and enable the temporal visualization of discrete gene regulatory events. The approach we present measures the rate of change of GFP fluorescence, which in turn reflects the rate of gene expression. We conducted fermentation and microtiter plates experiment using protein synthesis inhibitors to illustrate the feasibility of this system. Additional experiments using the classic gene regulation of the arabinose promoter demonstrate the utility of GFP as real time indicator of transcriptional regulation. With repetitive induction and repression of the arabinose promoter, GFP shows a cyclical change in fluorescence emission during culture.

Evidence of Lateral Phase Separation in Lecithin-Cholesterol Membranes
Gregory M. Troup, Steven P. Wrenn
Drexel University

Formation of cholesterol rich domains within the membrane has been reported, but conclusive evidence and a fundamental understanding of the domains are lacking. Cholesterol-rich domains if present might play an important role in phenotypic modulation during early atherogenesis. The goals of this investigation are to test the hypothesis that cholesterol-rich domains form via lateral phase separation in lecithin-cholesterol membranes, and to perform a systematic study to characterize the composition and temperature ranges over which such domains form. If cholesterol-rich domains exist it should be possible to melt them away by raising the temperature of the system. A fluorescent analog of lecithin was introduced into the membrane, and the fluorescent emission spectrum was recorded as a function of temperature and cholesterol loading.

High Pressure Dissociates Tailspike Protein Aggregates and Promotes Native Structure Formation
Brian G. Lefebvre and Dr. Anne Skaja Robinson
University of Delaware

Protein aggregation plays a major role in medicine, biochemical research, and in the biotechnology industry. We are using a number of techniques to study the reaction rates, and intermediates formed, during protein aggregation in order to understand and control this process. One of these techniques involves the application of hydrostatic pressure. Hydrostatic pressure can dissociate oligomeric proteins and other macromolecular complexes without denaturing the secondary and tertiary structure of the subunits, and has been used to determine the thermodynamics of assembly reactions. P22 tailspike protein is the model system for protein aggregation, as it forms aggregates under physiological conditions. Additionally, the structure is known, and tailspike intermediates along the folding and aggregation pathway have been characterized by native gel electrophoresis and size-exclusion high-pressure liquid chromatography. Tailspike protein aggregates formed during refolding were exposed to pressures between 15,000 and 35,000 psi. Pressure dissociated the aggregates, resulting in increased amounts of monomeric and dimeric folding intermediates, as well as native trimer, relative to ambient controls. Pressure also affects the intermediate conformations, as visualized by mobility shifts on native gels. These results show that hydrostatic pressure can convert protein aggregates to native protein without using chaotrophs, extremes of pH, or dilutions required in typical refolding procedures. To develop a molecular understanding of the aggregation mechanism, atomic force microscopy (AFM) was used to study macroscopic aggregate formation as a function of time. AFM images show that tailspike aggregates can adopt two morphologies: spherical clusters with diameters on the order of 100 nm and fibers up to 1 m in length. Section analyses performed on the spherical clusters indicate that they are composed of smaller units of roughly the same size as the tailspike folding intermediates. Ultimately, we will develop a mechanistic model that accounts for the macroscopic observations and the molecular behavior of the aggregates.

The use of a green fluorescent protein fusion tag to develop cellular systems for optimal expression of membrane proteins
Ronald Niebauer, Anne Skaja Robinson
University of Delaware

G-protein coupled receptors (GPCRs) are an important class of transmembrane proteins that mediate cellular response to diverse stimuli. Many diseases have been linked to GPCR function but little is known about expression, folding, and interactions of these proteins. In order to elucidate structural and functional properties, we plan to develop cellular systems for efficient expression of functional protein. Green fluorescent protein (GFP) will be used as a fluorescent fusion tag to identify the intracellular location and bottlenecks of the proteins. GFP is ideal for analysis because it requires no additional substrate and generally does not affect the native structure of the protein. The effect of chaperones and foldases can be studied once the GFP-GPCR fusion is complete. This marker will also aid in the development of methods to refold or improve the solubility of the protein.

Measurement of Intracellular pH Response of CHO Cells to Varying Culture Conditions Using BCECF
Daniel DeSantis, Amy Fritzinger, Jared Simons, Hong Sang, and Raj Mutharasan
Department of Chemical Engineering, Drexel University

The intracellular pH (pHi) response of Chinese Hamster Ovary (CHO) cells, growing in pseudo-continuous culture, to varying cultivation conditions was measured using the fluorescent probe BCECF through two different methods. Temperature, shear stress, and BCECF concentration are three parameters that were studied using the instantaneous pHi measurement method developed previously at Drexel University. The goals of these studies are to determine the lowest quantifiable concentration of BCECF that can be used to yield accurate pHi measurement results; the effect of shear stress due to bioreactor agitation speed on the pHi; and the pHi as effected by varying cultivation temperatures. A secondary method, perfusion measurement, was also used to determine both the effect of temperature shock, as well as the effect of varying cultivation temperatures.

Production of Therapeutic Proteins In Plants: Creation of a Plant Baculavirus System
Jason Collins, Edgard Carvalho, Wayne Curtis
The Pennsylvania State University

A plant tissue culture based system is being developed as a platform to produce proteins in sufficient quantity for testing and characterization purposes. In this plant tissue based expression system, a vector carrying the gene of interest is cloned into Agrobacterium tumefaciens. The bacterium and plant tissue are co-cultured, and the bacteria transfer the gene into the plant tissue. The plant tissue transiently expresses the foreign protein at high levels, and is then harvested. A proposed refinement includes causing the bacteria to transfer to the plant tissue a sub-genomic virus carrying the gene of interest. The plant tissue culture will be engineered to contain the viral amplification functions. It is anticipated that this system will produce higher protein yields due to the ability of the virus to transfer the foreign gene to additional cells beyond those initially transfected by the Agrobacteria. Additionally, auxotrophic bacteria could be used to diminish the effects of bacterial overgrowth on the plant tissue culture. Initial data will be presented to show the increase in transient expression due to mechanical wounding of the plant tissue, and also the pH dependence of transient expression when using acetosyringone to activate the Agrobacteria for gene transfer.

The Role of Cysteins and Disulfide Bonds on the Protein Folding of P22 Tailspike
Brenda Danek and Anne Robinson, University of Delaware, Newark, DE 19716
danek@che.udel.edu (302) 831-6697

Understanding how large, complex oligomeric proteins fold and assemble has been a goal of protein chemists for decades. A model system for investigating the assembly of these complex oligomers is the tailspike protein of the P22 bacteriophage, a homotrimer composed of 666 amino acids and eight cysteine residues per monomer chain. The macroscopic folding pathway of tailspike has been well studied, and monomer, dimer and a disulfide-bonded protrimer have been identified as folding intermediates. The disulfide-bonded protrimer is of particular interest as there are no disulfide bonds in the final, native structure. It is this feature that prompts the investigation into redox optimization of tailspike folding. The three cysteine residues toward the carboxyl terminus, at positions 496, 613 and 635, have been shown to exhibit sulfur reactivity, indicating that these are the most likely candidates to be involved in disulfide bonding during the folding reaction. Single serine mutants at each of these residues were expressed and purified from E. coli. In vitro characterization of these mutants, in trimer form, demonstrate similar stability to wild type tailspike when treated with various concentrations of guanidine chloride, as monitored by intrinsic tryptophan fluorescence. In addition, the single mutants have native-like circular dichroism spectra. However, significant differences were observed between the single mutant and wild type folding reactions under both low protein concentrations, which favor productive folding, and high concentrations, which lead toward aggregation. The single mutants fold two to five times slower than wild type under productive folding conditions, but this difference is even greater at aggregating conditions, where the appearance of trimer is more than fifty times slower than wild type. When two single mutants are mixed together in a single reaction, folding under aggregating conditions is significantly improved, with trimer appearing only two to four times slower than in the wild type reaction.

Investigation and Characterization of Gene Dosage on Expression of Archaeal b-glucosidase in the Yeast Saccharomyces cerevisiae
Jason D. Smith and Anne Skaja Robinson,
University of Delaware

Archaeal enzymes have great potential for industrial use, however expressing them in their natural hosts has proven challenging. Fermentation conditions for many archaea are far beyond modern fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae. Therefore, our goal is to bypass direct fermentation of archaea by overexpressing their enzymes in the yeast S. cerevisiae. Our model protein is the tetrameric b-glucosidase from the hyperthermophilic Pyrococcus furiosus. We engineered the b-glucosidase to be secreted into the medium and have shown successful secretion of active b-glucosidase. The effect of gene copy number on b-glucosidase secretion was studied and a maximally secreting transformant was found to secrete approximately 1 mg/L in batch culture. All transformants retained large intracellular fractions of b-glucosidase with an average approximately 370 times that of supernatant b-glucosidase values. Interestingly, our findings indicate a putative secretory bottleneck between the Golgi and the plasma membrane. We are currently investigating the nature of the bottleneck and developing cellular engineering solutions.

Deformation-Enhanced Fluctuations of the Erythrocyte's Spectrin-Actin Nodes and Relation to Spectrin Unfolding
James C-M. Lee and Dennis E. Discher
Bioengineering/ University of Pennsylvania
Philadelphia, PA 19104

To assess local elasticity in the red cells spectrin-actin network, nano-particles were tethered to actin nodes and their constrained thermal motions tracked. Cells were immobilized as well as controllably deformed by aspiration into a micropipette. Since the network is well-appreciated as soft, thermal fluctuations even in an unstressed portion of network were expected to be many 10s of nanometers based on simple equipartition ideas. Real-time particle tracking indeed reveals such motions for 40-nm fluorescent beads either tethered to actin directly within a cell ghost or else tethered to actin from outside a cell via glycophorin. Moreover, the elastically constrained displacements are significant on the scale of the networks inter-nodal distance of ~6080 nm. Surprisingly, along the aspirated projection where the network is axially extended by as much as two-fold or more fluctuations in the axial direction are increased by almost two-fold relative to motions in the unstressed network. A simple analytical model introduces the idea that this unexpected softening in the highly extended direction reflects forced unfolding of spectrin repeats, or else rapidly reversible dissociation within or between spectrin oligomers, and/or more subtle contributions from actin protofilament rotations.

Detachment of S. aureus from Collagen under Fluid Shear Conditions with the Aid of a Monoclonal Antibody
Lisa Mascari and Julia M. Ross
University of Maryland, Baltimore Campus

Receptor mediated adhesion of bacteria to biological surfaces is a significant step leading to infection. Novel methods to block these specific interactions have gained considerable interest due to an increase in bacterial antibiotic resistance. However, these methods are only useful as preventive strategies and not treatment strategies against infection. Recently, monoclonal antibodies specific for the S. aureus collagen receptor demonstrated the ability to displace attached cells from collagen in static assays. In this study, we report S. aureus detachment from collagen in a fluid shear environment aided by monoclonal antibodies that posses this displacement functionality. A parallel plate flow chamber was employed to create a defined shear environment for various levels of physiological shear (100-2300 s-1). The dynamic detachment of cells from collagen was quantified using phase contrast videomicroscopy and digital image processing in real time. Results demonstrate that detachment depends on S. aureus strain, mass transfer of the antibody to the receptor, and shear rate. We hypothesize that in regions of low shear (100-300 s-1) both antibody mass transport and shear force affect detachment rate, in regions of moderate shear (300-1500 s-1) detachment is effected only by antibody mass transport, and in regions of high shear (=2000 s-1/) detachment is minimized.

Affinity Ultrafiltration: Effects of ligand binding on Selectivity and Process Optimization
Jonathan Romero, Andrew Zydney
University of Delaware, Dept. of Chemical Engineering

The effective design of affinity ultrafiltration processes using a selective macroligand requires a detailed understanding of the effects of ligand binding interactions on product yield and purification. Theoretical calculations were performed to evaluate the performance of affinity diafiltration separations with both competitive and independent binding interactions for the product and impurity. The intrinsic selectivity for independent binding decreased during the diafiltration due to the increase in fractional impurity binding as the impurity is selectively removed. The opposite behavior was seen for competitive binding since the strongly bound product displaces the impurity from the binding sites. Purification-yield diagrams were used to examine the effects of affinity ligand concentration and binding constants on the separation. Model calculations were in excellent agreement with experimental data from single-stage and 2-stage cascade membrane systems for the separation of tryptophan isomers using bovine serum albumin as the stereoselective macroligand. Simulations with a fixed number of diavolumes show a clear optimum in product yield and purification factor at an intermediate ligand concentration due to the competing effects of the intrinsic selectivity and the rate of impurity removal. These results provide an appropriate framework for the design and optimization of affinity ultrafiltration systems.

The Effects of pH and Ionic Strength on Carbon Dioxide Transport Dynamics in Biorecators
Kenneth L. Urish and Wayne R. Curtis
Department of Chemical Engineering, The Pennsylvanis State University, University Park PA, 16802

The level of dissolved carbon dioxide is a key component in cell culture systems. In large-scale animal cell culture, dissolved carbon dioxide can quickly reach toxic levels and result in lower cell density and culture yield. In tissue culture, carbon dioxide is an important signal that can control metabolism and development. The mass transfer of carbon dioxide in culture is also a valuable measurement used in measuring the respiration quotient and can be used to predict the mass transfer rates of oxygen. Compared to the other gas components in a culture, carbon dioxide is more complex because it undergoes a series of reactions to form the inorganic carbon system. A mathematical model has been developed which predicts the dynamic response of a bioreactor to changes in the partial pressure of carbon dioxide using a proton balance. The model was used to investigate the effects of different culture variables on the mass transfer of carbon dioxide and dynamics of the inorganic carbon balance. The system was found to be extremely sensitive to pH and mildly to non-ideality introduced by the media's ionic strength. This has important implications for understanding the dynamics of dissolved carbon dioxide for measuring respiration quotients and using CO2 mass transfer to measure O2 mass transfer.

Calculating the mass transfer coefficients of oxygen from carbon dioxide mass transfer measurements
Kenneth L. Urish and Wayne R. Curtis
Department of Chemical Engineering, The Pennsylvanis State University, University Park PA, 16802

Accurate measurements of the oxygen mass transfer coefficient (KLa) are critical for successful scale up of bioreactors and controlling the metabolism of cultured cell lines. The measurement of the oxygen KLa usually involves stripping oxygen from the media of a reactor and then measuring the rate at which the dissolved oxygen returns (gas in * gas out technique). Problems with this method include the inaccuracy and unreliability of the dissolved oxygen probe and damage incurred to the cell culture during oxygen deprivation. A new method has been developed to measure the KLa of oxygen based on the bioreactor's inorganic carbon balance and the transport rates for carbon dioxide. It has been calculated and experimentally verified that the ratio of the KLa of carbon dioxide and oxygen is relatively constant, and the KLa of oxygen can be indirectly calculated from the KLa of carbon dioxide. The KLa of carbon dioxide can be experimentally measured from the bioreactor's response to a step change in carbon dioxide partial pressure to indirectly give the oxygen KLa.

Design of Smart Carriers for Peroral Protein Drug Delivery
Robert J. Murray, Mariko Morishita, Marc C. Torjman, Jeff I. Joseph and Anthony M. Lowman
Drexel University

The development of oral delivery systems for protein- and peptide-drugs has received significant attention. Two major problems exist in the development of such systems. First, significantly proteolytic degradation of the proteins and peptides occurs drugs in the stomach and upper portion of the small intestine. Also, the transport of the macromolecular drugs across the lining of the GI tract does not occur readily without the aid of absorption enhancers. To overcome these problems, we have developed a pH-responsive carrier that provides delivery to the small intestine as well as enhances gastrointestinal uptake of the proteins. In this study, graft copolymers of polymethacrylic acid and polyethylene glycol (P(MAA-g-EG)) were prepared and used as carriers of insulin. Due to the formation/dissociation of interpolymer complexes, the hydrogel will hinder diffusion in acidic environments and will protect the insulin from powerful digestive enzymes. At the higher pH conditions of the small intestine, these complexes will dissociate, resulting in polymer swelling and insulin release. The aims of this study were to investigate the release kinetics of insulin in vitro as well as the pharmacokinetics following in situ administration of the insulin loaded polymer in the GI tract.

Methods to study Controlled Drug Release in the Vitreous Humor of the Eye
Hyuncheol Kim(1,2) , Robert Lutz(2) , Martin Lizak(3) , Rupak K. Banerjee(2) , Michael Robinson(3) , Nam Sung Wang(1)
1 : University of Maryland, College Park Department of Chemical Engineering College Park, MD 20770
2: Division of Bioengineering and Physical Science National Institute of Health Bethesda, MD 20892
3: National Eye Institute National Institute of Health Bethesda, MD 20892

Ocular diseases such as retinitis, glaucoma, lymphoma, and macular degeneration are difficult to treat with systemic drug therapy because many drugs fail to deliver significant amounts of drug to deep eye structures. Intravitreal drug injections have been used with some success, but repeated injections for chronic treatment are impractical for most patients. With advances in controlled-release technology, interest has increased in the development of long-term, drug-releasing ocular implants for treating chronic eye diseases. Accurate determinations of drug concentrations at the various regions within the posterior chamber of the eye is crucial to effective drug delivery by the implants. The purpose of this work is to devise improved methods for monitoring drug levels in the eye non-invasively models that can predict these drug concentrations. To validate the measurement techniques and to improve our understanding of the mechanisms of intravitreal drug transport, model systems of the drug delivery are used. An in vitro mechanical model of the eye is used to study the relative importance of diffusion and convection of drugs through the vitreous humor. Drug movement is simulated by a marker dye moving through an agarose gel. Dye concentration is monitored optically with special imaging software. Once characterized, this in vitro model is used to validate the MRI methods for monitoring drug transport using contrast-enhancing agents such as gadolinium and its conjugates as drug surrogates. Additionally, mathematical finite element models are used to predict spatial and temporal drug levels in the chambers of the eye. These three-dimensional models incorporate characteristic of diffusion and convection as determined by in vivo MRI measurements.

Reactor Design for Submerged Root Culture: Role of Aeration and Tissue Morphology on Hydrodynamics
Lia D. Tescione and Wayne R. Curtis
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802.

Root cultures possess the ability to synthesize chemicals (secondary metabolites) with complex stereoisomer structures that may be difficult to produce by traditional chemistry. In addition, plant tissue culture is being explored as an alternative production system for eukaryotic, recombinant proteins. These classes of compounds are of interest to the pharmaceutical, food, or specialty chemicals industries. Commercialization of root cultures as alternative chemical production systems will depend on the feasibility of their large-scale cultivation. At large scales, in vitro root cultivation becomes limited by delivery of oxygen to the tissue. Various chemical reactor designs have been proposed for growing root cultures at large scales. In this work, one of these reactor configurations - the bubble column reactor - has been used at a pilot-scale (15 liters) to investigate the oxygen delivery limitations that cause decreases in growth and tissue respiration rates in liquid-submerged, sparged root culture systems. In an effort to understand the oxygen transport mechanisms in these systems, overall oxygen transport rates were measured in the pilot-scale bubble column reactor by gas-phase analysis, along with mixing of the bulk gas and liquid phases through use of tracer studies. The effects of aeration rate on liquid mixing, gas holdup, and tissue respiration rates were examined with two different root lines (Solanum tuberosum and Hyoscamus muticus). Mixing and apparent mass transfer rates were not enhanced by increasing aeration rates. The role of gas phase channeling in offsetting increased superficial gas velocities is discussed. Morphological tissue characteristics on bulk mixing were also examined using a hairless analogue developed for H. muticus (Bordonaro and Curtis, B&B, 2000). The elimination of root hairs proved to be effective at improving liquid mixing times and decreasing the extent of static gas in the bubble column reactor. These improvements were accompanied by 40% greater biomass accumulation and 20% higher respiration rates.


Abstracts Withdrawn

Multi-element Array Impedemetric Immuno-biosensors
Calvin Walker Jr., Anthony Guiseppi-Elie
Center for Bioelectronics Biosensor and Biochips