Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins. Conventional purification processes are lengthy, technically complicated, product specific and time-consuming. To address this issue, herein we develop a one step purification system that due to the nature of the non-selective secretion system and the versatility of ion-exchange membrane chromatography can be widely applied to the production of many recombinant proteins. This was achieved through the integration of the intrinsically coupled upstream, midstream and downstream processes, a connection that is rarely exploited. A bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography medium while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange medium, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level of 487 U/L/OD600, with more than 90% was localized in the extracellular medium. Both, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. Furthermore, formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. iv At harvest, the whole non-clarified culture broth was applied directly to a tangential flow filtration anion-exchange membrane chromatography system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry). Due to their size, cells remained in the retentate while the extracellular medium penetrated the membrane. Most contaminate proteins were captured by the anion-exchange membrane, whereas the purified PAC was collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), process yield (74%), and productivity (41 mg/L) was obtained.

In this study, a new bacteriocin isolated from a Leuconostoc mesenteroides subsp. cremoris strain was purified to homogeneity by pH mediated cell adsorption-desorption, solid phase extraction with Amberlite XAD-16, cation-exchange chromatography and hydrophobic interaction chromatography. The purification resulted in an electrophoretically pure protein that was smaller than 6 kDa as judged by SDS-PAGE. The bacteriocin was found to be very hydrophobic and cationic and could be adsorbed by synthetic calcium silicate due to its cationic and especially hydrophobic nature. It was observed that this bacteriocin was sensitive to &amp / #945 / -amylase in addition to proteinase K, trypsine, pepsine and chymotrypsine and had a bactericidal mode of action with a concomitant cell lysis. The results indicated that bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was more stable to combined effect of pH and heat than nisin, lacticin 481, lacticin 3147 and lactococcin G and was bactericidal between pH 2.0-12. It was found that the bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was stable to organic solvents and could be extracted with chloroform containing solvents efficiently for purification. The bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris was found to have a strong inhibitory activity against Listeria innoqua, Listeria monocytogenes, Bacillus cereus, Enterococcus faecalis, Lactobacillus delbrueckii, Lactobacillus cremoris, other Leuconostoc meenteroides strains and gram-negative bacterium Pseudomonas fluorescens. Some of the insensitive bacteria were observed to be sensitive when high concentration of the bacteriocin was used.

Hepatitis B virus (HBV), which belongs to the family Hepadnaviridae, is responsible for acute and chronic hepatitis. The vaccines presently used to immunize patients against HBV are recombinant subunit vaccines consisting of viral surface antigens (S protein). However, they are expensive and their use is limited in poor countries. For that reason, HBV remains an important worldwide health problem. Of the 2 billion people who have been infected with the HBV, more than 350 million have chronic (lifelong) infections, who face increased risk of developing cirrhosis and hepatocellular carcinoma. In this study, high-level expression of recombinant Hepatitis B surface Antigen (rHBsAg), PreS2-S was achieved in the methylotrophic yeast, Pichia pastoris. For this aim, a single copy of HBV M gene (PreS2-S) was inserted at the downstream of the alcohol oxidase (AOX1) promoter of the pPICZA vector. rHBsAg protein could then be expressed intracellularly by induction with methanol. High cell density fermentation was followed by chromatographic separation to obtain pure rHBsAg. Humoral response after immunization with the purified protein was observed in mice using commercial Hepatitis B surface antigen kits. It was verified by the atomic force microscopy that rHBsAg has been produced in the desired conformation.

In this study, we evaluated the potential of water-in-oil microemulsions for the separation of beta-galactosidase (lactase) from other proteins. The ability of beta-galactosidase to break down the milk carbohydrate lactose gives the enzyme considerable commercial importance. The extent of solubilization of a commercial Kluyveromyces lactis preparation of beta-galactosidase into microemulsion droplets formed from 200 mM bis (2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane was measured as a function of buffer type, pH, ionic strength, and protein concentration. Our results showed that, due to the large molecular weight of beta-galactosidase (MW~ 220-240 kDa, dimeric form), the enzyme was taken up by the microemulsion droplets mainly under very low salt conditions. Based on these results, we designed a one-step separation procedure, in which a small volume of aqueous buffer containing the protein mixture is added to an organic surfactant solution. Microemulsion droplets form in the oil and capture protein impurities of smaller molecular weights, while excluding the high molecular weight target protein. This causes the beta-galactosidase to be expelled into a newly formed aqueous phase. The feasibility of this one-step process as a bioseparation tool was demonstrated on a feed consisting of an equal mixture of beta-galactosidase and the test protein beta-lactoglobulin. Recovery and separation of the two proteins was analyzed as function of buffer type, pH, ionic strength, and protein concentration. Results showed that separation was most complete at 100 mM KCl salt concentration, where the droplets were big enough to carry beta-lactoglobulin but too small for lactase. At 100 mM salt concentration, we recovered 92% of the total lactase activity in a virtually pure form. The same separation scheme was then tested on crude extract obtained from a cell culture broth of the yeast Kluyveromyces lactis. Cells of the yeast K. lactis were disrupted by minibeadbeater, forming a crude extract that was used as the feed in our separation process. A 5.4-fold purification factor of the extract was achieved, with 96% activity recovery. The results showed our one-step separation process to be an interesting method for the production of beta-galactosidase as a technical enzyme: it has the potential to achieve a continuous, large-scale partial purification of the enzyme, potentially reducing the number of steps required in downstream process.

Food poisoning is mainly caused by pathogenic microorganisms and is now a severe problem worldwide. Therefore, rapid and sensitive methods are required to detect foodborne pathogens. A locally isolated bacterium, Bacillus licheniformis B38 was selected for this study. The spores of B. licheniformis B38 were induced by Schaeffer’s sporulation medium containing KCl, MgSO4.7H2O, Ca(NO3)4, MnCl2 and FeSO4. Schaeffer-Fulton endospore staining was used to differentiate spores and vegetative cells, where spores were stained green and vegetative cells were stained red. In order to separate the spores from the cells, a two-phase system was used to obtain pure spore suspension for following experiments. Spore coat proteins were extracted by SDS-8 M urea sample buffer and visualized by two different types of coomassie brilliant blue staining solutions. One of the staining solutions was more suitable for gel elution by diffusion. An ~10 kDa spore coat protein was selected for protein purification. Based on the given results, the protein purification by liquid chromatography was less convincing than using gel elution by diffusion technique. The two hypothetical protein sequences, P06552 and P45693, from the ~10 kDa spore coat protein were identified. In the preliminary study of B. licheniformis B38 spores detection by surface plasmon resonance, several binding parameters were studied. Dot blot was done to verify the reaction between the Bacillus spores polyclonal antibody against the B. licheniformis B38 spore coat protein. The most promising result was the binding of 0.1 mg/mL polyclonal antibody (analyte) to the 0.2 mg/mL spore coat protein at pH 2 (ligand) which showed 5.74 RU. The differences between a dot blot and a SPR detection techniques are described.

Η ακρίβεια και η πιστότητα της διαδικασίας διπλασιασμού του γονιδιώματος είναι μείζωνος σημασίας για την κυτταρική επιβίωση. Τυχόν ανωμαλίες όπως μεταλλάξεις ή χρωμοσωμικές ανωμαλίες είναι δυνατόν να οδηγήσουν στην εμφάνιση κακοήθειας ή σε πρόωρο κυτταρικό θάνατο. Τα παραπάνω συνηγορούν στη μεγάλη σημασία που έχει η άρτια λειτουργία και ρύθμιση του κυτταρικού κύκλου. Δύο πρωτεϊνες, οι geminin και cdt1, κατέχουν πολύ σημαντικό ρόλο κατά τη διαδικασία ρύθμισης του κυτταρικού κύκλου. Πιο συγκεκριμένα, η cdt1 αποτελεί έναν βασικό παράγοντα αδειοδότησης της αντιγραφής. Δρα συνεργατικά με την πρωτεϊνη Cdc6 προκειμένου να γίνει η πρόσδεση του εξαμερούς συμπλόκου MCM2-7 στο προ-αντιγραφικό σύμπλοκο (pre-RC), διασφαλίζοντας με τον τρόπο αυτό τις απαραίτητες συνθήκες για τη διαδικασία αδειοδότησης της αντιγραφής (Maiorano D. et al., 2000). Η geminin συνιστά τον φυσικό αναστολέα της cdt1 στα μετάζωα. Προσδένεται ισχυρά στην τελευταία μετά την έναρξη της σύνθεσης του DNA, παρεμποδίζοντας με αυτόν τον τρόπο την επαναπρόσδεσή της στο προ-αντιγραφικό σύμπλοκο (Tada S. et al., 2001; Wohlschlegel J.A. et al., 2000). Η διαδικασία αδειοδότησης της αντιγραφής παρουσιάζει ανωμαλίες σε περιπτώσεις καρκινικών κυττάρων. Πιο συγκεκριμένα, έχει δειχθεί σταθερή υπερέκφραση της cdt1 σε καρκινικές κυτταρικές σειρές, τόσο σε πρωτεϊνικό όσο και σε μεταγραφικό επίπεδο (Xouri G. et al., 2004). Παρομοίως, αυξημένη έκφραση της cdt1 έχει παρατηρηθεί και σε περιπτώσεις καρκίνου του παχέος εντέρου καθώς και πρώιμου καρκίνου του πνεύμονα, στον άνθρωπο (Bravou V. et al., 2005; Karakaidos P. et al., 2004). Τα παραπάνω αποτελέσματα έχουν προκύψει κατόπιν μελέτης, η οποία πραγματοποιήθηκε στο εργαστήριό μας. Αναφορικά με τη geminin, αυξημένα επίπεδα έκφρασής της έχουν συσχετιστεί με καρκινώματα παχέος εντέρου καθώς και με τη διαίρεση κακοηθών κυττάρων, γενικότερα (Gonzalez M.A. et al., 2005; Wohlschlegel J.A. et al., 2002). Επιπροσθέτως, η geminin βρίσκει εφαρμογή ως ανεξάρτητος δείκτης σε περιπτώσεις επιθετικού καρκίνου του μαστού (Gonzalez M.A. et al., 2004). Βασιζόμενοι στα παραπάνω, θεωρούμε ότι το σύμπλοκο geminin-cdt1 συνιστά έναν ιδανικό στόχο για το σχεδιασμό νέων αντικαρκινικών φαρμάκων. Το πρώτο βήμα προς αυτήν την κατεύθυνση αποτελεί ο μαζικός έλεγχος συνθετικών συστατικών, τα οποία να έχουν την ικανότητα διάσπασης του συμπλόκου. Ενδεχόμενη εύρεση τέτοιων συνθετικών συστατικών καθώς και μετέπειτα φυσικοχημική βελτιστοποίησή τους είναι δυνατόν να οδηγήσει στη δημιουργία ενός νέου αντικαρκινικού φαρμάκου. Η ολοκλήρωση της χαρτογράφησης του ανθρώπινου γονιδιώματος συνέβαλε στην ταυτοποίηση νέων πρωτεϊνικών μορίων στόχων, τα οποία εμπλέκονται στο μοριακό μηχανισμό διαφόρων ασθενειών. Με βάση τις εξελίξεις των τελευταίων χρόνων στον τομέα της φαρμακοβιομηχανίας, η αξιοποίηση αυτής της γνώσης συνδέεται με το μαζικό έλεγχο συνθετικών συστατικών έναντι αυτών των μορίων στόχων. Απώτερο στόχο αποτελεί η αναγνώριση κατάλληλων συνθετικών συστατικών τα οποία θα έχουν την ικανότητα να αλληλεπιδρούν με το μόριο-στόχος και κατ’επέκταση να μεταβάλλουν τον τρόπο λειτουργίας του, προκειμένου να έχουμε το επιθυμητό φαρμακολογικό αποτέλεσμα. Αποφασιστικής σημασίας στην παραπάνω διαδικασία, αποτελεί η σωστή επιλογή της κατάλληλης μεθόδου μαζικού ελέγχου των νέων υποψήφιων φαρμάκων – συνθετικών συστατικών – έναντι του μορίου στόχου. Στην παρούσα διπλωματική, επιλέχθηκε προς εφαρμογή η μέθοδος FRET. Ένα από τα βασικά της πλεονεκτήματα είναι η υψηλή αναλογία ‘σήματος-θορύβου’ που εμφανίζει καθώς και η υψηλή ποιότητα των δεδομένων που παρέχει. Αν και αποτελεί μία σχετικά καινούρια τεχνική, εντούτοις αποτελεί μία από τις πιο βασικές μεθόδους της σύγχρονης φαρμακοβιομηχανίας λόγω της αξιοπιστίας που την χαρακτηρίζει και επιπλέον της συμβατότητάς της με αυτοματοποιημένες τεχνικές. Ασφαλώς, απαραίτητη προϋπόθεση για την εφαρμογή της συγκεκριμένης μεθόδου αποτελεί η απομόνωση της υπό μελέτη πρωτεϊνης. Η έκφραση του πρωτεϊνικού συμπλόκου geminin-cdt1 πραγματοποιήθηκε με τη χρήση βακτηριακών συστημάτων έκφρασης ετερόλογων πρωτεϊνών. Επίσης, ο καθαρισμός του συμπλόκου υπήρξε επιτυχής και βασίστηκε στην εφαρμογή των τεχνικών της χρωματογραφίας συγγένειας και της χρωματογραφίας διήθησης σε πηκτή. Το επόμενο βήμα ήταν να διαπιστώσουμε εάν η μέθοδος FRET καθιστά δυνατή την ανίχνευση σχηματισμού του συμπλόκου. Πράγματι, κάτι τέτοιο ήταν εφικτό καθώς σε συγκέντρωση 60-80nM του συμπλόκου, παρατηρήθηκε αύξηση του σήματος σχεδόν κατά πέντε φορές υψηλότερα από το επίπεδο “θορύβου”. Το αποτέλεσμα αυτό είναι ιδιαίτερα σημαντικό καθώς συνεπάγεται ότι με τη συγκεκριμένη μέθοδο είναι εφικτός ο μαζικός έλεγχος συνθετικών συστατικών, τα οποία θα έχουν την ικανότητα διάσπασης του συμπλόκου. Οι λόγοι που συντελούν στην καταλληλότητα της μεθόδου για αυτό το σκοπό έγκεινται αφενός στην ευαισθησία την οποία εμφανίζει (αύξηση του σήματος μέχρι και πέντε φορές) και αφετέρου στην ειδικότητα και την αξιοπιστία της, όπως έχει δειχθεί και με τα αντίστοιχα πειράματα. Σημαντικό επίσης πλεονέκτημα αποτελεί και η μικρή σχετικά ποσότητα πρωτεϊνης (60-80nM), η οποία απαιτείται. Σύμφωνα με τα δεδομένα που έχουν προκύψει από την παρούσα διπλωματική , το FRET assay συνιστά μία ιδανική μέθοδο για την πραγματοποίηση μαζικού ελέγχου συνθετικών συστατικών, τα οποία θα έχουν την ικανότητα διάσπασης του συμπλόκου geminin-cdt1. Δεδομένης της πολύ ισχυρής αλληλεπίδρασης του συγκεκριμένου συμπλόκου, πραγματοποιήθηκαν μεταλλαγές σε τρία υψηλά συντηρημένα κατάλοιπα της cdt1, τα οποία κατέχουν κυρίαρχο ρόλο στην αλληλεπίδραση με τη geminin, σύμφωνα με κρυσταλλογραφικά δεδομένα (Lee C. et al., 2004). Οι μεταλλαγές αυτές ενδέχεται να συμβάλλουν σε ένα πολύ πιο εύκολα διασπάσιμο σύμπλοκο, γεγονός που μπορεί να οδηγήσει στην ευκολότερη και γρηγορότερη ταυτοποίηση υποψήφιων συνθετικών συστατικών. / The accurate and timely duplication of the genome is vital for cell survival. Mutations rearrangements or loss of chromosomes can be detrimental to a single cell as well as to the whole organism, causing malignant cell growth or death. Origins of replication are licensed by a multi-subunit complex (pre-replicative complex: pre-RC) during G1 (Lei M & Tye B.K., 2001). Pre-RC assembly is an ordered, sequential process in which the Origin Recognition Complex (ORC) first binds to each replication origin and then recruits two other proteins: Cdc6 and Cdt1 (Bell S.P. & Dutta A., 2002). These two proteins function synergistically to load the six mini-chromosome maintenance helicase proteins (MCM2-7) onto the pre-RC, establishing the conditions for DNA licensing (Maiorano D. et al., 2000). The prevention of ectopically induced re-replication is accomplished by functionally redundant mechanisms, including sequestration of MCM by Crm1 (Yamaguchi R. & Newport J., 2003), inactivation and export from the nucleus of Cdc6 (Delmolino L.M. et al., 2001; Jiang W. et al., 1999; Pelizon C. et al., 2000) and degradation of Cdt1 (Nishitani H. et al., 2001). Metazoans, have evolved an additional system for preventing re-replication: Geminin, which was originally identified in Xenopus as essential factor to exit from mitosis (McGarry T.J. & Kirschner M.W., 1998), binds tightly to Cdt1 and prevents Cdt1 assembly onto pre-RC (Tada S. et al., 2001; Wohlschlegel J.A. et al., 2000). Licensing system members are misregulated in cancer cells and differential expression of licensing components could be used for the diagnosis and prognosis of cancer (Shreeram S. & Blow J.J., 2003). Over-expression of Cdt1 can predispose cells to a malignant transformation. It has been shown that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level (Xouri G. et al., 2004). Moreover, Cdt1 protein is highly expressed in cases of human colon cancer and primary human lung carcinomas (Bravou V. et al., 2005; Karakaidos P. et al., 2004). Similarly, Geminin is also over-expressed in colon carcinomas and its expression levels were directly related to the cellular proliferation index in proliferating malignant cells (Gonzalez M.A. et al., 2005; Wohlschlegel J.A. et al., 2002). Furthermore, expression of Geminin is considered as an independent indicator of adverse prognosis in cases of invasive breast cancer (Gonzalez M.A. et al., 2004). Given the crucial role of the Geminin-Cdt1 complex in cell cycle regulation and cancer, this complex could serve as target for the discovery and development of novel anti-cancer drugs. This requires the screening of compounds that are capable of disrupting the geminin-cdt1 complex. For that purpose we performed a HTP (high-throughput)-compatible assay, called FRET-assay. The acronym FRET stands for Fluorescence Resonance Energy transfer. The principle of the assay is based on the radiationless transfer of energy from an excited donor fluorophore (Europium Cryptate) to a suitable acceptor fluorophore (XL665). The first step was the expression and purification of the geminin-cdt1 complex. The complex was expressed by using E. coli bacterial cells and purified by metal affinity chromatography on a Ni+2 column. As soon as the complex was ready to use, we next tried to investigate whether the formation of the complex was detectable by using the FRET assay. Indeed, at the complex concentration of 60nM and 80nM, the signal was about 5 times above background. This was a first indication that the Geminin-Cdt1 complex can be used successfully for energy transfer based assays. Given the very high binding affinity of the two proteins (Lee C. et al., 2004), it could be quite unlikely to find a compound that can disrupt the complex. To overcome that obstacle, three single mutations were made at the highly conserved Geminin-contacting residues of hCdt1, Y170, F173 and L176. The mutation of these residues to alanine can possibly provide a more easily disruptable complex, which could be of importance concerning the faster and easier identification of any candidate compounds. Our data suggest that hGeminin-Cdt1 complex can be considered as a promising target for compound screening. Given the high importance of Geminin-Cdt1 balance for maintaining genomic stability integrity and that both proteins have been correlated with cases of cancer, that screening could hopefully lead to the discovery and development of lead compounds towards anti-cancer drugs.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.

Parasites of phylum Apicomplexa cause devastating diseases on a global scale. Toxoplasma gondii, the etiological agent of toxoplasmosis, and Plasmodium falciparum, the most virulent agent of human malaria, have the most substantial effects on human health and are the most widely studied. The success of these parasites is due in part to a sophisticated molecular arsenal that supports a variety of novel biological processes including a unique form of host cell invasion. Accessing the protective environment of the host cell is paramount to parasite survival and is mediated through an active invasion process: the parasite propels itself through a circumferential ring known as the moving junction (MJ) formed between its apical tip and the host cell membrane. The MJ ring is comprised of a parasite surface protein (AMA1) that engages a protein secreted by the parasite into the host cell and presented on the host cell surface (RON2). Thus, through an intriguing mechanism the parasite provides both receptor and ligand to enable host cell invasion. Prior to the studies described herein, the characterization of the AMA1-RON2 association was limited to low-resolution experiments that provided little insight into the functional and architectural details of this crucial binary complex. Towards elucidating the mechanism of AMA1-RON2 dependent invasion, I first structurally characterized T. gondii AMA1 bound to the corresponding binding region of RON2; analysis of the AMA1-RON2 interface along with biophysical data revealed an intimate association likely capable of withstanding the shearing forces generated as the parasite dives through the constricted MJ ring. To investigate the role of the AMA1-RON2 complex across genera, species and life-cycle stages, I next characterized the AMA1-RON2 complex from a distantly related genus within Apicomplexa (Plasmodium) and from a divergent pairing within T. gondii. By combining structural, biophysical and biological data, I was able to generate a detailed model describing the role of AMA1 and RON2 in MJ dependent invasion, which is currently supporting efforts to develop novel vaccines and cross-reactive small molecule therapeutics. / Graduate / 0487 / tonkin.ml@gmail.com

A platform for vaccine development has been developed at Georgia State University utilizing recombinant Salmonella typhimurium flagellin (FliC) fused to an antigen that can be overexpressed in Escherichia coli grown in a two-stage fermentation. The flagellin acts as an adjuvant to increase the immunopotency of the fused antigen. Flagellin is the ligand for Toll-like Receptor 5 (TLR5), a part of the innate immune system. Binding of the flagellin:antigen recombinant protein to TLR5 triggers a strong innate and adaptive immune response to the fused antigen leading to a potentially strong protective immunity to the antigen. Purification of the recombinant FliC fusion protein must meet rigorous criteria in order to be used as a vaccine. One of the major issues in purifying recombinant proteins expressed in a Gram-negative bacterium is the removal of endotoxin. Small amounts of endotoxin present in a vaccine can lead to serious complications, including death. Recombinant proteins are also expressed as either soluble or insoluble protein when over expressed in E. coli. Soluble proteins expressed by the bacterium are properly folded and biologically active, however removal of contaminants such as endotoxin, can be problematic. Insoluble protein is improperly folded and biologically inactive. The insoluble proteins aggregate into inclusion bodies with little or no contaminants associated with the protein, making purification easier. However, in order to restore the biological activity of the insoluble protein, it must first be solubilized and then refolded. This process is often expensive and time consuming, as there is currently no standardized method for protein refolding. In this study a purification method for the soluble protein of two FliC constructs, full-length FliC and FliC fused to a Marburg virus antigen, was evaluated for effectiveness in purification, removal of endotoxin and maintaining TLR5 activity. The proteins of interest were purified utilizing only the soluble protein containing the properly folded and biologically active recombinant protein. Utilizing methods for purification that take advantage of physical and chemical properties of the protein the recombinant proteins were purified and the level of endotoxin reduced to levels acceptable for use as a vaccine. The TLR5 activity of the soluble recombinant proteins was compared to recombinant protein that had been purified using a denaturing and refolding step. The soluble protein elicited a higher TLR5 response at a lower concentration of protein than the refolded protein. Purification of the soluble fraction also involved fewer step and less time than purification of both the soluble and insoluble protein.