UMBC Links
UMBC
College of Engineering
Chemical and Biochemical Engineering (CBE)
Biochemical Engineering Graduate Students (BioChEGS)

Abstracts

Keynote Presentation
Oral Presentations
Poster Presentations

Oral Presentations

A. Improved sindbis viral expression systems for mammalian cell culture
Toey Nivitchanyong¹, Yien Che Tsai², Michael J. Betenbaugh¹, and George A. Oyler^2
1Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218
²Department of Neurology, University of Maryland, Baltimore, MD 21201 

     The Sindbis virus has been engineered to allow the expression of heterologous genes encoded on the virus.  The use of such viral expression systems enables the rapid production of high levels of recombinant protein in mammalian cell culture. Unfortunately, the Sindbis virus is lethal to most cells in culture, limiting this expression system to transient production.  However, if the expression system could be engineered to limit the lethality inherent in the Sindbis virus, the potential exists to enable long term, sustained expression of recombinant proteins by mammalian cells infected with the virus. Such a system may include modifications to the virus or the cells themselves in order to reduce the cytopathic effects.  Non-cytopathic replication competent viruses of 2 Sindbis viral strains, TE and 633, were developed via point mutations in order to obtain persistent heterologous gene expression in Sindbis-infected cell lines.  The virus encodes for the expression of a model green fluorescent protein (GFP) which is being expressed in infected Baby Hamster Kidney (BHK) cells and Chinese Hamster Ovary (CHO) cells.  Cells infected with non-cytopathic virus were able to recover after infection, while cells infected with normal virus died within 3 days.  Cells infected with non-cytopathic virus also expressed slightly higher amount of GFP, in comparison to the normal virus.  In addition, we explored modifications to the host BHK and CHO cells themselves as another method for limiting the cytopathic effects of the Sindbis virus.   We have engineered BHK and CHO cells to stably express these anti-apoptotic genes: Bcl-2 and Bcl-2D.  The combination of cell lines expressing anti-apoptotic genes and non-cytopathic virus produced synergistic effect in terms of viability and GFP production.  The rate of cell death post infection was delayed, the cells recovered, and in some cases cells proliferated and continually expressed GFP after several passages.  The GFP production was increased several fold, in comparison to infection with normal virus.  Such combinatorial strategies may lead to the generation of viral expression systems that provide high level production of heterologous proteins in mammalian cells of biotechnological interest.

B. engineering yeast cells for optimal expression of membrane  proteins
Ronald T. Niebauer and Anne Skaja Robinson
Department of Chemical Engineering, University of Delaware, Newark DE

     The G-protein coupled receptors (GPCRs) are an important class of transmembrane proteins that mediate cellular response to diverse stimuli. Many diseases including cancer and heart disease have been linked to GPCR function. GPCRs represent the target for the majority of presently prescribed pharmaceutical drugs, but little is known about expression, folding, and interactions of these proteins. The goal of this research is to understand the cellular interactions limiting expression and use this information to develop a Saccharomyces cerevisiae system for high-level expression of functional GPCRs. A specific GPCR, the human adenosine receptor, was tagged with the green fluorescent protein reporter and was determined to be functional based on ligand binding assays and correctly localized to the plasma membrane based on confocal microscopy. However, the net foreign protein production rate appeared to decrease over time although the corresponding mRNA levels remained relatively high and the unfolded protein response was not activated. Taken together our data suggest that a translational or post-translational bottleneck is limiting expression. Current strategies for maximizing expression include identifying cellular interactions with the GPCR and optimizing expression conditions.

C. SCALE-UP OF TRANSGENIC TOBACCO CELLS THAT EXPRESS INTIMIN OF ENTEROHEMORRHAGIC ESCHERICHIA COLI O157:H7 FOR USE AS AN ORAL VACCINE IN CATTLE
¹Kristin M. O’Neill, ¹Anne M. Schilthuis, ¹Calvin A. Leiter, ¹Kurt M. Neihaus, ²Nicole A. Judge, ²Edda Twiddy, ²Alison D. O’Brien, and ¹Wayne R. Curtis
¹Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802-4400
²Department of Microbiology and Immunology, F. Edward Herbert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799.

     Intimin is the primary adhesin of enterohemorrhagic Escherichia coli O157:H7, a pathogen carried by cattle and transmitted to humans via food and contaminated water. In a matter of months production was scaled up from small shake flasks to a pilot scale reactor resulting in the production of 1.3 Kg dry weight of transgenic plant cells.  This material, expressing the C-terminal 261 amino acids of intimin (Int261), was generated for use in “proof of principle” studies to demonstrate the ability to develop an oral bovine vaccine against pathogenic E. coli.  Both a stirred-tank bioreactor (60 L) and a less capital-intensive oxygenated carboy culture are shown to be feasible means of generating this scale of transgenic plant biomass.  A control strategy of incremental increase in gas flow rate, combined with oxygen supplementation was used to provide oxygen delivery at a minimum gassing rate.  Antibiotic selection pressure was not required over a 13-week period needed to maintain Int261 expression during scale-up.  Extended media autoclave times of up to 90 minutes used for bioreactor sterilization had only minimal impact on nutrient uptake, growth and intimin expression. Plant tissue was transformed, produced and available for feeding studies in a fraction of the time required to develop and grow transgenic plants.

D.  Effect of dilution rate on plasmid productivity
Alok Asoor
Department Of Chemical Engineering, Villanova University, Villanova, PA 19085.

     In this study, the effects of dilution rate on cellular growth and plasmid production for a continuous fermentation of Escherichia coli (E.coli) pUC 18 were investigated. The results of the empirical study were used to develop a mathematical dynamic process model for a continuous fermentation system. This strain of bacteria has a plasmid with a gene that codes for ampicillin resistance so ampicillin was used as a selective pressure. The bacterium was allowed to grow in a batch mode until it reached a stationary phase and then continuous mode of operation was initiated. Samples for optical density and DNA analysis were collected at regular time intervals. The results show that the cell density values go down as the dilution rate is increased. Also the cells shed their plasmid within a few hours into the continuous mode. Other possibility is that, at the end of the batch the culture is rich with plasmid containing cells and so there is a huge amount of β–lactamase. When the continuous mode of operation is initiated, with ampicillin coming in at a low concentration, most of it is destroyed by the β - lactamase already present in the culture. So the cells shed their plasmids because they don’t need to produce any more β–lactamase and the culture is plasmid free. 200mg/l of ampicillin is not enough to kill the plasmid free cells as the threshold limit is very high compared to 200 mg/l. Further recommendation is that the batch culture be spiked with ampicillin some time before switching to continuous mode. This would get rid of all β–lactamase and a proper selective pressure could be maintained

E. REDUCING OUTER MEMBRANE PERMEABILITY BARRIER IN WHOLE CELL BIOCATALYSIS
Ye Ni, Rachel R. Chen, and Xuan Guo
Department of Chemical Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA 23284

     In many applications, whole-cell biocatalysts are preferred. But cell envelopes often constitute permeability barriers to substrates. Whole-cell catalyzed reactions are generally 10-100 fold slower than isolated enzyme-catalyzed reactions. Our research aims to accelerate whole-cell biocatalysis by reducing membrane permeability barrier using molecular engineering approaches. E. coli cells with genetically altered outer membrane structures were used in the study. Specifically, a lipopolysaccarides mutant and a murein lipoprotein mutant were combined with periplasmically expressed enzymes. The reduction of outer membrane permeability barrier by genetic methods led to significant increase (up to 380%) of reaction rates of whole-cell catalyzed reactions. Notably, the mutation in outer membrane can render the outer membrane completely permeable to substrates, a barrier-less condition that maximizes the reaction rate. Moreover, the extent of increase in biocatalysis rate was found dependent on the substrates and the nature of mutations introduced in the outer membrane structure.

F. SELF-CLEAVING AFFINITY TAGS RE-ENGINEERED FOR HIGH-    THROUGHPUT APPLICATIONS
Judy F. Hsii¹, Lydia M. Contreras², and David W. Wood¹. (1) Department of Chemical Engineering, Princeton University, Princeton, NJ 08544 (2) School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14850

     The cloning, expression and purification of industrially relevant proteins is a tedious, time-consuming, multi-step process.  A recent improvement has been the development of self-cleaving affinity tags for the rapid and simple purification of arbitrary product proteins.  At the same time, conventional cloning methods have also been simplified through the development of one-step topoisomerase-based cloning and site-specific recombinant cloning.  A combination of these new technologies promises a major advance in high-throughput protein production and purification.  To accomplish this goal, three new self-cleaving affinity tags were engineered to include DNA sequences required for one-step cloning.  The result is an efficient, rapid and general method for PCR amplified genes to be cloned and the encoded proteins expressed and purified.  Preliminary results indicate that this method will find applications in proteomics, directed evolution, drug discovery and many other areas.  

G. INFLUENCE OF SYMBIOTIC SPECIES INTERACTIONS ON TRANSCRIPTIONAL RESPONSE IN HYPERTHERMOPHILIC MICROORGANISMS
Matthew R. Johnson, Clemente I. Montero, Shannon B. Conners, Keith R. Shockley, Stephanie L. Bridger, and Robert M. Kelly
Department of Chemical Engineering, North Carolina State University, Raleigh, NC 27695-7905

     Species interaction in high temperature environments have not been well characterized to this point, and information about transcriptional response to the establishment of a symbiotic coculture have yet to be explored.  In this study, both pure and co-cultures at 80°C of model hyperthermophilic microorganisms, Methanococcus jannaschii and Thermotoga maritima, have been used to explore high-temperature microbial ecology from several perspectives. When grown in coculture, the archaeon M. jannaschii, an autotrophic methanogen, utilizes hydrogen and carbon dioxide produced from the bacterium T. maritima as its sole source of hydrogen and carbon. In turn, T. maritima benefits from the presence of the methanogen due to the removal of inhibitory extracellular hydrogen, resulting in a four-fold increase in T. maritima maximum cell density. 
     Using the available genome sequence, a full-genome cDNA microarray for T. maritima was constructed and used to examine gene expression during log phase in replicate cultures containing T. maritima cells, cultured with and without M. jannaschii. Gene expression data for T. maritima cells grown alone and under co-culture conditions were analyzed using two separate ANOVA mixed models, a global normalization model and a gene-specific model. Results have indicated that many of the bottlenecks and stresses of growing with hydrogen limitation are removed by both the coculture and sparging cases. Major differences occur though between the sparging cases in regards to ABC transporter and carbohydrate active enzyme inventory usage, suggesting exopolysaccharide-based biofilms may be used in the coculture environment.  Cross species hybridization was also examined by hybridizing RNA extracted from pure cultures of M. jannaschii against the T. maritima microarray.  Among the issues investigated were possible mechanisms for intra- and interspecies signaling, factors leading to biofilm formation, motility regulation and coupled metabolic features, including interspecies hydrogen transfer, under varying population dynamics such as species presence and phase of growth.

H. Detection of Pathogen E.coli 0157:H7 Using Monoclonal Antibody Immobilized Piezoelectric Glass Microcantilever
Gossett A. Campbell and Raj Mutharasan
Drexel University, Chemical Engineering Department, Philadelphia, PA 19104

     Self-excited microcantilevers can be used to measure small mass changes in the order of picograms.  The purpose of this study is to examine their applicability in detecting pathogens.  Method: The Lead Zirconate Titanate/Glass (PZT/Glass) unimorph microcantilevers (2 – 4 mm length) were fabricated and their mechanical resonance spectra characterized. An alternating electric field applied across the PZT layer causes polarization in the direction of the applied field which results in expansion and contraction of the PZT material. This piezoelectric effect sets the sensing tip (glass) in oscillation and resonance is determined by measuring phase angle. The resonance frequency is proportional to the inverse square root of mass change at cantilever tip. The cantilever tip was silanized with a reactive alpha amine end. Using zero length crosslinker molecules EDC and N-Sulfohydroxy succinamide (Sulfo NHS), monoclonal antibody (MAb) specific to the target pathogen E.coli 0157:H7 (EC) was covalently immobilized. Selectivity of the biosensor to EC was demonstrated by experiments conducted under various contaminating conditions. The cantilever dynamics were modeled using a two dimensional Navier model. Results: EC solutions at various concentrations (7 x 10² to 7x10⁶ #/mL) and mixed with wild strains (total concentration: 7x10⁶ #/mL) were exposed to MAb-immobilized cantilever.  Frequency shifts of  194, 214, 328 and 536 Hz were obtained at concentrations of 700, 7x10⁴ , 7x10⁶ , and  7x10⁷  #/mL EC atttachment, respectively.  Resonant frequency shifts of 1370, 501, and 0 Hz were obtained for EC contaminated with a wild strain at 0%, 50% and100%, respectively.. Note, two different cantilevers were used to carry out the two sets of experiments. The mass sensitivity of the cantilevers were 9.1x10^-8 and 3.2x10^-9 grams per hertz, respectively. Energy Dispersive Spectroscopy was used to confirm the presents of EC on the sensing glass surface. This technique showed high levels of phosphorous and sulfur which is associated with cellular material. Verification of attachment was also obtained by exposing the EC-attached microcantilevers to low pH buffer which realeased the attached antigen, and resulted in increased resonance frequency back to the original value. Eigenvalue analysis of the cantilever dynamics agreed well with experimentally determined resonance in air. Conclusions: The results indicate that EC concentration at a low value of 700 #/mL can be detected with good sensitivity. The model gave good prediction of the natural resonant frequency of the sensor in air and  provides the means a priori for fabrication of a cantilever for which specific characteristics of resonance frequency and quality factor are desired. Acknowledgement: EPA R82960401 (funding), Prof Wan Shih (use of Impedance Analyzer), Dan Luu (Data Acquision Program).

I.  PROTEOME ANALYSIS TO EVALUATE PHYSIOLOGICAL CHANGES IN ESCHERICHIA COLI EXPOSED TO SIMULATED NON-IDEAL MIXING
Babu Raman, M. P. Nandakumar, Vignesh Muthuvijayan, and Mark R. Marten
Department of Chemical and Biochemical Engineering,
University of Maryland Baltimore County (UMBC), Baltimore, MD 21250

     Non-ideal mixing often occurs in production scale fed-batch fermentations, and can lead to gradients in both oxygen and carbon concentration. This has often been cited as one of the main reasons for performance discrepancies on scale-up. In this study, we simulated non-ideal mixing conditions during fed-batch fermentation and used proteome analysis to determine how this affected the bacteria. Simulated poor mixing conditions were achieved by repeatedly pulsing glucose in 180s cycles (90s on/90s off) in carbon limited fed-batch fermentation (20L). Intracellular proteins from constant feeding phase (control) and pulse feeding phase were separated using two-dimensional polyacrylamide gel electrophoresis and 78 protein spots showing significant difference in expression (>2-fold) between the two conditions were identified using MALDI-TOF mass spectrometry. We validated our proteome analysis methods by comparing protein expression between exponential phase and fed-batch phase cells, and our results were consistent with literature on cellular responses to carbon limitation. Under pulse feeding, up-regulated proteins included amino acid biosynthetic enzymes (13), translational apparatus related proteins (10), proteins involved in amino acid transport (3), and stress & regulatory proteins (5). Under constant feeding conditions, carbon catabolism (4), transport and binding (8), and central intermediary metabolism (4) proteins were up-regulated. This implies that cells experienced increased stress under pulse feeding, and maintained an excess of translational machinery and amino acid biosynthetic capacity, possibly, to aid in rapid growth when nutrients became available. We suppose such knowledge would ultimately lead to strategies for improving the performance of industrial fermentations.

Poster Presentations

1. A Novel Protein Purification Technology: One-step Affinity Purification Using Simple Centrifugation
Mahmoud Reza Banki and David Wood
Department of Chemical Engineering, Princeton University, Princeton, NJ 08544

     A cost effective, environmentally friendly, and scaleable protein expression and purification system is presented.  This is an economic alternative to conventional affinity purification using expensive resins or affinity beads.  The process starts with a triplet fusion precursor protein consisting of an affinity tag, a recently developed engineered self-cleaving element and a product protein is expressed in E.coli.  The fusion is then purified via the affinity of the fusion tag to an economical, biodegradable polymer by simple centrifugation, without the addition of conventional affinity reagents.  Subsequently, affinity tag self-cleavage is triggered by temperature or pH to release the native, highly-purified product protein from the polymer, without conventional column processing or enzymatic affinity tag removal.  The cleavage reaction is irreversible, hence, pH and temperature can be reverted back to optimal conditions for product protein activity following cleavage.  Purification results including yield and activity for three proteins: maltose binding domain, NusA, and β-lactamase will be presented. Although we have focused on these three proteins thus far, this method is not limited by the properties of the product protein.  The critical advantage of this process is that following expression, a native protein can be purified from a cell lysate simply by brief centrifugation and temperature or pH induced product protein release, eliminating all chromatographic and enzymatic treatment steps which have so far prohibited upscaling of such a process to industrial levels. In addition, this purification technology is applicable to proteins expressed not only in bacteria, but also other organisms for efficient production of large amounts of high-purity proteins and enzymes. 

2.  Alginate Strings with Genetically Engineered Fibroblasts and their Application in Spinal Cord Regeneration
Saravanan Kanakasabai⁺, M Murray^#, I Fisher^#, and MA Wheatley^*+
School of Biomedical Engineering, Science and Health Systems, Department of Neuroanatomy^#, College of Medicine, Drexel University, Philadelphia, PA 19104

     We have optimized a method for producing a multifunctional graft that would aid in spinal cord regeneration following injury. The graft comprises of strings of alginate bioconjugated with a laminin pentapeptide that aids in neuronal cell adhesion. These strings are 400-500 μm in thickness and could be bundled to act as a graft at the site of injury. In vitro studies have shown that rat (NB2a) and human (SHSY5Y) neuroblastoma cell lines adhere to, and differentiate on these modified alginate strings. The strings also have the capacity to hold genetically modified fibroblasts that release neurotrophins that would aid in neuronal regeneration. Staining studies show that these genetically engineered cells are viable inside the graft and are secreting the required neurotrophic factor. The strings are also coated with specific polyamines that prevent host immune reaction against these cells. These strings are strong enough to be surgically transplanted to the spinal cord, and may ultimately help in bridging the gap at the injury site. The graft promotes neuronal cell adhesion and regeneration thereby acting as a physical bridge across the site of injury.

13. How much is too much: The effect of Adsorption on protein stability in Hydrophobic Interaction chromatography
Yunzhi Xiao, Alexander S. Freed, John P. O’Connell, and Erik J. Fernandez
Department of Chemical Engineering, University of Virginia

     The stability of proteins can be threatened by adsorption on hydrophobic interaction chromatography (HIC) surfaces. HIC has become an important tool in downstream processing, and the ability to predict the effect of salts and additives on both adsorption and stability is critical to rapid and effective process development as well as design of new chromatographic media. In this research we describe experimental analysis of α-lactalbumin stability on HIC media by hydrogen exchange-mass spectrometry. We have also developed some simple theoretical methods for interpreting the combined effects of salts on additives on both chromatographic retention and stability. The model relationships help explain the observed trends, clarifying the mechanism by which they act. Further, their ability to semi-quantatively describe the observed behavior may provide simple relationships or ultimately detailed design tools to facilitate process development.

14. IMPROVING NEURON-TO-ELECTRODE SURFACE ATTACHMENT OF PC 12 CELLS FOR AN IMPEDIMETRIC WHOLE CELL BIOSENSOR
Gymama E. Slaughter, Erhard Bieberich¹, Gary E. Wnek² and Anthony Guiseppi-Elie^2,3
Center for Bioelectronics, Biosensors and Biochips, School of Engineering, Virginia Commonwealth University, P.O. Box 843038, 601 West Main Street, Richmond, VA 23284-3038.
¹Institute of Molecular Medicine and Genetics, School of Medicine, Medical College of Georgia,1120 15^th Street Room CB-2803 Augusta, GA 30912.
²Department of Chemical Engineering and ³Department of Emergency Medicine, Virginia Commonwealth University, P.O. Box 8430328, 601 West Main Street, Richmond, VA 23284-3028

     The w-amine alkanethiols, cysteamine (CA) and 11-amino-1-undecanethiol (11-AUT), were adsorbed as self assembled monolayers (SAMs) onto 250mm gold microelectrodes that were microlithographically fabricated within 8-well cell culture plates and investigated as a means to improve neuron-to-electrode surface adhesion.  Cell adhesion proteins, collagen, fibronectin and laminin, were covalently coupled to the aminoalkanethiol decorated gold electrodes via acid-amine hetero-bifunctional cross-linking. Using FITC-tagged laminin, confocal fluorescence microscopy of both CA and 11-AUT SAM-modified and non-modified gold microelectrodes confirmed coupling of the protein to the electrode and was readily distinguishable from non-specifically adsorbed protein. Dynamic Contact Angle (DCA) measurements of laminin physisorbed directly onto gold or covalently immobilized via CA or 11-AUT SAM had similar (θ_a=63° – 65° and θ_r=7° - 9°) contact angels. Tapping mode AFM of these protein-bearing surfaces showed dimerized protein aggregates of similar surface roughness. Confluent layers of PC12 cells cultured on both non-modified and SAM-modified gold microelectrodes were examined by A.C. impedance (50 mV p-t-p at 4kHz). The CA SAM-modified surfaces were identified as being best suited for optimal neuron-to-electrode surface attachment using laminin. The impedimetric responses of the effect of exogenous agents on PC12 cells were examined on CA SAMs covalently derivatized with laminin surfaces.  Impedimetric responses of PC12 cells that undergo calcium exocytosis and ion modulation in the presence of calcimycin, nifedipine, mannitol, carbachol, NGF, dexamethasone and forskolin were obtained. Our results demonstrate that a change in electrophysiological behavior, such as exocytosis or modulation of ions, is detectable using temporal impedance monitoring.

15. In silico reconstruction of nutrient-sensing signal transduction pathways in Aspergillus nidulans
Vignesh Muthuvijayan and Mark R. Marten
Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, Baltimore, MD 21250

    All the organisms respond to changes in the environmental conditions such as nutrient availability, oxygen limitation, pH variation, osmotic stress etc. Any environmental change is accompanied by physiological changes in the living organisms. The cascade of processes by which an extra-cellular signal interacts with a receptor at the cell surface, causing a change in the level of a second messenger and ultimately effects a change in the cell’s functioning is called signal transduction. Different environmental changes trigger a variety of signal transduction pathways which is required for the physiological adaptation of the cell. In this work, we have used bioinformatic techniques for understanding the probable nutrient-sensing signal transduction pathways in Aspergillus nidulans, model filamentous fungi. For this, we performed sequence homology studies on the genomes of Aspergillus nidulans and Saccharomyces cerevisiae. The Basic Local Alignment Search Tool program “blastp” was used to study the homology of proteins. The results showed that most of the yeast signal transduction genes have significant homologues in Aspergillus nidulans indicating that the signal transduction pathways in both these fungi would probably be highly similar. Based on this, the probable pathways in Aspergillus nidulans were constructed using the well-established models in Saccharomyces cerevisiae.

16. INVESTIGATION OF CELL STRESS DURING HETEROLOGOUS PROTEIN EXPRESSION USING A GREEN FLUORESCENT PROTEIN STRESS SENSOR
Ping Xu, Francis J. Doyle III^†and Anne Skaja Robinson
Departments of Chemical Engineering, University of Delaware and ^†University of California at Santa Barbara

     Heterologous protein expression can easily saturate the cell’s capacity to properly fold protein and result in a cellular response known as the unfolded protein response (UPR). Since the UPR has been implicated in low protein yields, we hope to gain an understanding of the dynamics of the UPR and the effect of this response on protein expression in order to optimize and control cellular fermentations. In this study, the yeast Saccharomyces cerevisiae was used to express a model single-chain antibody (scFv) protein. Single-chain antibodies are of great interest as potential therapeutic and diagnostic agents, but can be difficult to express recombinantly, and have been shown to activate the UPR in yeast. The green fluorescent protein (GFP) was used as a molecular sensor to detect UPR activation in the cell. The cellular chaperone Binding Protein (BiP) has been shown to be a key player in UPR activation, since a decrease in BiP levels can activate the UPR signaling cascade. Here we examined the role of BiP in the onset and duration of the UPR. However the overexpression of BiP did not relieve the stress of scFv expression. Another protein, Protein Disulfide Isomerase (PDI) acts as chaperone, isomerase and oxidase in yeast. Although our experiments showed the overexpression of PDI as well as BiP increased the secretion of scFv level, the GFP stress sensor indicated that only PDI relieved the stress of scFv expression. The reasons were found that BiP bound scFv and improved the synthesis rate of scFv, but could not fold scFv into its active form; on the contrary, PDI could fold scFv properly. Our model is that BiP and PDI cooperated in the folding of scFv, and PDI catalyzed the formation of the disulfide bonds of scFv, enabling faster folding, so that BiP could be released and inactivate the UPR. 

17. INVESTIGATION OF THE MECHANISM OF SMALL HEAT SHOCK PROTEIN β–AMYLOID FIBRIL FORMATION AND TOXICITY PREVENTION
Sungmun Lee¹, Kenneth Carson², Allison Rice-Ficht², and Theresa Good³.
¹Chemical Engineering, Texas A&M University, College Station, TX, USA,
²Medical Biochemistry and Genetics, Texas A&M University, College Station, TX, USA,
³Chemical and Biochmeical Engineering, UMBC, Baltimore, MD, USA.

     β-Amyloid (Aβ) is a major protein component of senile plaques in Alzheimer’s disease, and is neurotoxic when aggregated. The size of aggregated Aβ responsible for the observed neurotoxicity and the mechanism of aggregation are still under investigation, however, prevention of Aβ aggregation still holds promise as a means to reduce Aβ neurotoxicity. In research presented here, we show that Hsp20, a novel β-crysatllin isolated from the bovine erythrocyte parasite Babesia bovis, is able to prevent aggregation of denatured alcohol dehydrogenase when the two proteins are present at near equimolar levels.  We then examined the ability of Hsp20 to prevent Aβ amyloid formation as indicated by Congo red binding, and found that not only was Hsp20 able to almost completely prevent Congo red binding, but it was able to do so at molar ratios of Hsp20 to Aβ of 1 to 1000.  Electron microscopy confirmed that Hsp20 does prevent Aβ fibril formation.  Hsp20 was also able to significantly reduce Aβ toxicity to both SH-SY5Y and PC12 cells at similar mole ratios.  Surprisingly, at higher concentrations of Hsp20, the protein no longer displayed its aggregation inhibition and toxicity attenuation properties.  This work could contribute to the development of a novel aggregation inhibitor for the treatment of Alzheimer’s disease.

18. INVESTIGATION OF STAPHYLOCOCCUS AUREUS BIOFILMS GROWN UNDER PHYSIOLOGICALLY RELEVANT SHEAR STRESS USING A PARALLEL PLATE FLOW CHAMBER
Patrick Ymele-Leki and Julia M. Ross
Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County

     Staphylococcus aureus is one of the most common causes of hospital- and community-acquired infections. S. aureus has been shown to colonize surfaces in organized biofilm communities, which are a common cause of chronic or persistent infections. Such biofilms may form on numerous indwelling medical devices, including central venous or urinary catheters, mechanical heart valves, and tampons. They are also capable to transiently colonize the nostrils, pharynx, or damaged skin surfaces of healthy adults. Moreover, data confirming the resistance of biofilm cells to antimicrobial agents continue to be generated. Little is known, however, on the mechanisms involved in the maturation of S. aureus biofilms under physiological conditions. We hypothesize that both the architectural organization and the protein expression of staphylococci biofilms will vary depending on the mode of growth. The goals of this research are to perform a comprehensive analysis of the structure, growth and maturation of Staphylococcus aureus biofilms under physiologically relevant shear conditions and to study the expression levels of known adhesion molecules on detached planktonic cells. Preliminary results use a parallel plate flow chamber to investigate the adhesion characteristics and biofilm growth patterns of S. aureus Phillips on collagen under a constant shear rate of 100-s^-1 maintained for a 24-hr period. Visual characterization of biofilm development is done in situ in real time with videomicroscopy using a phase-contrast microscope. Understanding the architectural and phenotypic properties of S. aureus biofilms under physiologically relevant flow conditions may lead to the development of novel therapeutic methods pertaining to the prevention and annihilation of chronic infections due to that organism.

19. NOVEL BIOSENSOR FOR HIGHLY SENSITIVE GLUCOSE MONITORING
Xudong Ge, Leah Tolosa, and Govind Rao
Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250

     Highly sensitive glucose monitoring has potential applications in many fields such as monitoring of glucose levels in LB medium or interstitial fluids. Here we describe a glucose sensor for highly sensitive glucose monitoring based on a dual-labeled glucose binding protein (GBP). The plasmid encoding the L255C variant of GBP was constructed through site-directed mutagenesis, and the protein was expressed in E. coli strain NM303. After it was purified, the engineered GBP was labeled with an environment-sensitive fluorophore, acrylodan, at the single cysteine mutation and a nonenvironment-sensitive fluorophore, ruthenium, at the N-terminal. The acrylodan emission is quenched in the presence of glucose while the ruthenium emission remains constant, thereby can be taken as a reference. The sensitivity of the sensor is in micromolar range. The effect of temperature on the response of the sensor was measured and analyzed. The enthalpy change for glucose binding calculated from the apparent binding constants is -42 kJ/mol. In addition to the ratiometric measurements, this dual-emitting GBP also allows for modulation-based measurements.

20. OPTIMIZATION OF EXPRESSION OF THE HYPERTHERMOPHILIC β-GLUCOSIDASE PROTEIN IN SACCHAROMYCES CEREVISIAE
Nicole E. Richardson and Anne S. Robinson
Department of Chemical Engineering, University of Delaware, Newark, DE 19716

     Proteins from hyperthermophilic organisms have great potential use as industrial enzymes, but currently cannot be efficiently produced due to extreme growth conditions in the native host. However, it has not proven easy to express these proteins at an economically feasible level in other systems. We have been studying the expression of β-glucosidase from Pyrococcus furiosus, an archaebacteria which thrives at extremely high temperatures, in the Saccharomyces cerevisiae system. We have chosen S. cerevisiae due to its compartmentalized secretion and folding, and the fact that it has a fully sequenced genome. The P. furiosus β-glucosidase is a well-characterized, extremely stable oligomeric protein which is representative of several proteins found in hyperthermophilic organisms. It previously has been shown that β-glucosidase expression is increased in S. cerevisiae at 37°C rather than 30°C, but cells fail to thrive at higher temperatures. We now report that the cells grown at 37°C have a lower unfolded protein response (UPR), are able to secrete protein for longer periods of time, up to 90 hours, and have higher intracellular levels of β-glucosidase during the course of 24 hours. This leads us to believe that the cells are utilizing different proteins at the higher temperature, and are finding some way to combat the stress caused by this additional protein expression.

21. PATTERN RECOGNITION DEPENDENCY ON ADSORPTION HEAT AND ACTIVATION ENERGY
Guy N. Tchoupo, Arvind K. Srivastava and Anthony Guiseppi-Elie
Center for Bioelectronics, Biosensors and Bioships, School of Engineering, Virginia Commonwealth University, Richmond, P.O. Box 843038, 601 West Main Street, Richmond, VA 23284

     Design of polymer-based, chemo responsive chemical sensors for a given set of target vapors is a challenging task as the mechanism of the sensing phenomena is still not well understood. There are various models available in the literature for gas sensing. The Langmuir adsorption isotherm and equation is one of the most commonly used models to explain adsorption kinetics for a range of gas-solid interaction. In this work, we proposed a novel approach to select gas-sensing materials for a given set of volatile organic compounds (VOC). We do so by varying some parameters (e.g. activation energy and heat of adsorption) in the Langmuir equation that directly relates to gas-solid interaction followed by neural network pattern recognition to evaluate to what extent variations in adsorption kinetics results in satisfactory adsorbate classification. For our simulation experiments data sets were generated by taking different combinations of activation energy of adsorption (Ea) and heat of adsorption (Q) for different gas-solid interaction at normal temperature and pressure (NTP). Data sets thus generated were used to build a neural network model to classify the vapors and estimate their concentration. Performance of the NN model was evaluated by perturbing Ea and Q until the classification accuracy was maintained within an acceptable range. This study will enable us to understand the degree of variations in sensor property that is tolerable for the identification and quantification of target analytes.

22. PHOTODYNAMIC THERAPY AS AN ALTERNATIVE TREATMENT FOR SOME CANCERS AND AUTOIMMUNE DISEASES
Jennifer Ruiz and Theresa A. Good
Chemical and Biochemical Engineering, University of Maryland Baltimore County (UMBC)

     Certain cancers, including adult T cell leukemia and cutaneous T cell lymphoma, as well as certain autoimmune diseases, are characterized by the abnormal expression of the a chain of the IL-2 receptor complex an the surface of T cells. The use of this abnormality to target disease associated T cells and selectively deliver toxins has been suggested and has proved successful in many cases. This study describes the use of monoclonal antibodies to deliver a photosensitizer, chlorine e₆ (Ce₆), to IL-2Ra bearing T cells, as a way to increase effectiveness and selectivity of photodynamic therapies. Selective destruction of leukemia T cells was achieved after photosensitizer internalization and activation with a He:Ne laser. Blocking the IL-2R with and anti-IL-2R monoclonal antibody showed a reduction of treatment efficacy, confirming the roll of this receptor in the delivery of Ce₆. However, attempts to block the receptor with IL-2 showed an increased efficacy; the reasons for this behavior are yet to be explained. The treatment was successfully applied to normal human T-cells, activated with lectins to express the IL-2R; the procedure proved to be more effective in killing this type of cell, probably due to the expression of the full IL-2R complex, which has a higher affinity for the monoclonal antibody carrier than the α chain alone. Currently, efforts are focus on elucidating the kinetics of IL-2R expression on the hope of developing a model for prediction of treatment dosage and effectiveness.

23. Protein C and Copper (II) Metal Ion Interfacial Interaction in Immobilized Metal Affinity Chromatography
James J. Lee and Duane F. Bruley
University of Maryland, Baltimore County (UMBC),Chemical & Biochemical Engineering Department, 1000 Hilltop Circle, Building ECS 304,Baltimore MD 21250

     Studies are being done to produce large quantities of low cost Protein C (PC) for the treatment and prevention of many potential pathological disease states as a result of blood clotting. Presently the most commonly used anticoagulant drug for PC deficiency is coumadin. This oral anticoagulant acts by regulating several blood clotting factors. Though often used for long-term therapy, coumadin is also likely to cause complications such as: minor to persistent bleeding, sensitivity reactions, and skin necrosis in more severe cases. It has also been suggested that long-term use of this oral anticoagulant could lead to tissue and organ damage. Reduced oxygen transport due to blood agglutination within the body results in tissue death and organ failure. Low cost PC production availability is therefore extremely important. PC can help achieve blood hemostasis in many dread disease conditions such as sepsis, cancer, HIV (AIDS), etc.
     Immobilized Metal Affinity Chromatography (IMAC) has high specificity and can be used for difficult separations among homologous, high molecular weight therapeutic proteins at relatively low cost to current methods, such as Immunoaffinity Chromatography. Prior research has demonstrated the effectiveness of IMAC for the purification of PC from blood plasma fraction Cohn IV-1. To produce a viable PC product, PC must be completely separated from other structurally similar blood factors. The similarity of these different therapeutic blood proteins makes the separation very difficult and expensive. The objective of our research is to study the interfacial phenomena of protein adsorption and desorption in IMAC. Molecular interactions within the chromatography column involve several essential parameters, which influence the protein’s physical characteristics that define how the protein interfaces and interacts with the metal ion. Biochemical and biomechanical studies of the chosen IDA–Cu/PC system in IMAC is based upon previous research. The biochemical study investigates the separation phenomena through thermodynamic measurements of adsorption and desorption. The biomechanical study uses a visualization program to examine and compare the structures of PC and other critical homologous blood factors.
     We are investigating the separation of PC from blood plasma fraction Cohn IV-1 as our chosen model system based on its availability, provided by the American Red Cross (ARC), and our knowledge of the protein. The interfacial interaction is very important to understand because it will allow for IMAC optimization for the purification of PC as well as other homologous therapeutic proteins. This will help determine the most effective processing conditions to achieve our goal to provide a technique that separates with high bioactivity, purity, and yield at low cost.

24. RAPID PRODUCTION OF HETEROLOGOUS PROTEINS VIA A PLANT- TISSUE-CULTURE BASED TRANSIENT EXPRESSION SYSTEM
Jason Collens and Wayne R. Curtis
Department of Chemical Engineering, Pennsylvania State University University Park, PA 16802

     A rapid method to generate proteins, such as therapeutics and diagnostics, in plant tissue would help make plants more competitive compared to traditional production platforms. We are developing a transient gene expression system in plant tissue culture to decrease the time required to produce a protein in plant tissue. An intron-containing GUS reporter gene is being used to determine optimal parameters for protein production. First, the bacterium Agrobacterium tumefaciens is engineered with the reporter gene. The bacteria transfer the reporter gene to the plant tissue during co-culture. The GUS protein is transiently produced in the plant tissue prior to the genes integration into the chromosome. The intron ensures that the functional reporter protein is only produced by the plant tissue, and not by the bacteria. A time course has shown increasing protein expression to approximately three days of co-culture. Agrobacteria will overgrow plant tissue in culture, which can result in low reporter gene expression or plant tissue death. An auxotrophic Agrobacterium strain was generated to reduce the stress of co-culture. This bacterium requires cysteine for normal growth and metabolism. The utility of this cysteine auxotroph is demonstrated through a co-culture with Nicotiana glutinosa cells, in which the plant cells co-cultured with the auxotroph expressed 85-fold more GUS than the cells co-cultured with the prototrophic bacteria. It is anticipated that increased protein production could be achieved if the plants could make additional copies of the gene coding for the protein of interest. We have generated and are testing transgenic plant cultures containing either an ethanol-inducible, copper-inducible, or constitutively expressed viral replication-associated gene. The reporter gene has also been modified to be amplified by this viral replication-associated protein. It is anticipated that the replication-associated protein will augment the number of reporter gene copies in the plant nucleus, increasing production of the protein of interest.

25. REFOLDING YIELD FROM LOW TEMPERATURE PRE-INCUBATION OF P22 TAILSPIKE PROTEIN
Junghwa Kim and Anne Skaja Robinson
Department of Chemical Engineering, University of Delaware, Newark, DE 19716

     P22 tailspike protein (TSP) was used as a model protein to investigate how large, multimeric proteins fold and assemble into the correct native structure. P22 tailspike protein is a trimer of identical 666 amino acid chains. One interesting property of tailspike structure is that the main part of each monomer is comprised of a beta helix domain. In the final structure, three parallel beta helices associate, and through intertwining of beta sheets, form the thermostable, SDS-, and protease-resistant tailspike trimer. Another interesting feature of TSP is that non-native disulfide bonding is involved in a productive folding pathway leading to native tailspike protein. To understand folding and assembly of tailspike protein, a refolding experimental method with ice pre-incubation was used to slow the overall protein folding kinetics and to accumulate and investigate folding intermediates efficiently. In this work, we found that ice incubation helped develop the beta helix domain of subunits as well as accumulation of disulfide- bonded intermediates, which are critical factors to drive correct assembly between subunits. Also, we observed the structural change of TSP in hierarchical orders toward native trimer. The improved refolding yield of TSP at 30°C after an ice incubation of at least 30 minutes supports the idea that these structural changes at 0°C favor productive folding.

26. SPATIALLY SELECTIVE BIOMOLECULE ASSEMBLY FOR SIMPLE AND ROBUST BIOLOGICAL DETECTION
Hyunmin Yi, Li-Qun Wu, Gregory F. Payne and William E. Bentley
Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD 20742

     Recent advances in biosensor technology have greatly expanded the scope of analytical methods in practical applications such as bioprocess monitoring, biological threat detection, medical diagnosis and food safety. Despite such advances, there has been relatively slow progress in and significantly increasing needs for development of robust biological/abiological interface to construct biosensors. We have developed a simple and robust biomolecule assembly procedure utilizing natural polysaccharide chitosan as such an interface material. By our protocol, chitosan's unique pH and electrostatically responsive properties have been fully exploited to generate a spatially organized chitosan layer onto conductive inorganic substrate surfaces that are routinely generated by photolithography. Next, chitosan's chemical reactivity enabled us to couple probe biospecies such as nucleic acids and proteins onto such generated layer in a target- and spatially- selective manner. This technique also allows sequential assembly of probe species without complex equipments or robotic facilities. Our presentation will emphasize recent advances that are part of our ongoing effort to multiple analyte detection in a microscale fluidic environment.

27. ULTRA LOW CONCENTRATIONS (70 counts/mL) OF E. COLI 0157:H7 IS DETECTED USING TAPERED FIBERS
Kishan Rijal and R. Mutharasan
Department of Chemical Engineering, Drexel University, Philadelphia, PA 19104

     Single mode continuous tapered fibers were fabricated with waist diameters of 3-8 microns and of various waist lengths (3 to 10 mm). The evanescent field generated in the tapered region was used to sense analytes placed in the waist region. The tapering results in increased access to the evanescent field, and thus the changes in transmitted light is influenced by extremely small changes in concentration of target analyte just adjacent to the fiber surface. In the present study, the tapered region of the fiber was silanized, and subsequently immobilized with antibody to E. coli 0157:H7 (EC) via a heterofunctional crosslinker, N-hydroxy succinimide. The antibody-immobilized taper was mounted on an optical bench and the tapered region brought in contact with EC suspension at various concentrations. Evanescent scatter at 469 nm was recorded by measuring transmitted light as a function of time. The change in transmitted light intensity provides for a signal proportional to cell concentration at the fiber surface and thus the identity and concentration of pathogen in the test sample. The tapered fibers were sensitive enough to detect the presence of EC at concentrations as low as 70 cells/ml. Antibody immobilized fibers were also able to differentiate the pathogen from a contaminating suspensions wild E. coli strain.

28. USING PROTEOME ANALYSIS TO UNDERSTAND STARVATION STRESS IN FILAMENTOUS FUNGI
Yonghyun Kim, M. P. Nandukumar, and Mark R. Marten
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore, MD 21250

     Starvation stress response in filamentous fungi is a complex naturally occurring process of enzymatic self-degradation and is an important topic to study as it complicates bioprocesses and has the potential to lower the yield of desired biotechnology products. In addition, a better understanding of the starvation stress response may facilitate the design of new antifungal compounds targeting increasingly resistant strains of pathogenic fungi. Currently there is little molecular understanding of this process in filamentous fungi. We hypothesize that the intracellular events involved are regulated by a group of regulatory kinase enzymes. In this study, proteome analysis will be used to capture the dynamics of both kinase activity and other proteins that are involved. As the complete genome sequence of Aspergillus nidulans is now available, two-dimensional gel electrophoresis (2DE) and in-gel kinase assay followed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry will help identify proteins differentially expressed at significant levels during starvation. Expression level-time profiles of expressed proteins will be categorized using cluster analysis, allowing us to deduce possible functions of proteins and helping us map the molecular events that accompany starvation.

29. USING THE FLOW CHAMBER TO OBSERVE VACUOLATION IN FILAMENTOUS FUNGI AS AN EFFECT OF LACK OF NUTRIENT
Judith Kadarusman and Mark R. Marten
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore, MD 21250

     Filamentous fungal fermentations are used to produce nearly $1 billion in industrial enzymes annually, yet many suffer from high broth viscosity, resulting in reduced productivity. Recently, we found that pulsed feeding during fed-batch fermentation led to smaller mycelia, reduced viscosity, and increased productivity. We propose that the cells’ response to pulse-feeding of nutrient in a similar to “starvation” (lack of nutrient). One way to test is to compare vacuolation in starved, pulse-fed, and continuous-fed cells. The goal in this study was to observe vacuolation as the cells respond to deprivation of a particular nutrient, glucose. A model fungus, Aspergillus oryzae, was grown in a narrow (~50 micron) gap, parallel plate flow chamber mounted on a microscope stage. Video microscopy was used to observe the mycelial growth over time, and image analysis techniques were used to quantify the changes in vacuolation, morphology, and other cellular degradation phenomena within mycelial elements. Preliminary results showed that vacuole formation increased in tip areas as cells lacked glucose. The formation of increased vacuoles affects the cells’ susceptibility to fragmentation.

30. WHOLE BLOOD STUDIES OF THE ROLE OF CLFA IN STAPHYLOCCAL-COLLAGEN BINDING INTERACTIONS
Michael A. Johnson and Julia M. Ross
Chemical and Biochemical Engineering, University of Maryland Baltimore County

     In staphyloccal blood-borne infections, bacteria and platelets often aggregate on the sub-endothelium where the extracellular matrix is exposed. In past studies the S. aureus adhesin, clumping factor A (ClfA), has been shown to be a critical virulence factor in several experimental models of infections. However, the extent of the role of ClfA in infections is uncertain. In this study, the interaction between ClfA and possible bridging molecules is studied under shear stress conditions in whole blood during the development of infected thrombi. The S. aureus binding interactions were evaluated using a series of mutant strains of S. aureus and the bacterial cell adhesion location (at the collagen surface or in the platelet aggregate) was measured using confocal laser microscopy. Results demonstrated S. aureus ClfA-binding interactions, likely mediated by fibrinogen, accounted for greater than 50% of bacterial binding in the platelet aggregate. Studies are underway to investigate the role of other S. aureus adhesin molecules that also bind fibrinogen. In conclusion, the role of ClfA should be considered when developing novel therapeutics for blood-borne infections.