Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfide-bond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white lysozyme (HEWL) antibody fragment was carried out at 37\ubaC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/l after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain

Utilization of various projectiles to mitigate fouling in tubular heat exchangers

Heat exchangers are the workhorse of most chemical, petrochemical, food processing and power generating processes. Of the many types of heat exchangers, approximately 60% of the market is still dominated by shell and tube heat exchangers. One major problem of heat exchangers and particularly the shell and tube type is directly related to the deposition of unwanted materials on the heat transfer surfaces. Fouling may cause one or more of several major operating problems: i) reduction of heat transfer, ii) under-deposit corrosion, iii) increased pressure loss and iv) flow mal-distribution. There are many different mitigation techniques available in the market to maintain the surface of heat exchangers clean to some extent. Among them are projectiles of various shapes, materials and hardnesses which circulate via a separate loop through the exchanger. The advantages of this method include effective fouling mitigation and stable operating conditions. Having said that, there are nevertheless numerous unanswered questions such as optimum injection interval, minimum required shear force to remove fouling layers, applicability of projectiles at elevated temperatures, minimum required velocity of projectile propulsion, and the criterion for the selection of projectiles for any specific fouling process. The present study, as part of a European Project entitled Clean-Ex, endeavors to address some of these questions. A test rig was designed and constructed to simulate conditions under which fouling occurs in water service processes. The rig includes an online cleaning device which enables introduction of projectiles for various operating scenarios including i) continuous or ii) at different time intervals. A comprehensive set of experimental runs was carried out for crystallization fouling of CaSO4 solutions with and without projectiles. Due to laboratory restrictions, fouling runs were performed at accelerated conditions to rigorously characterize the impact of projectile cleaning in terms of injection intervals and various types of projectiles. The experimental results showed that the projectiles are capable of removing parts of the fouling layer at the early stage of the fouling process. The cleaning efficiency decreases as the fouling layer builds up such that the projectile is not effective when the asymptotic fouling is approached. In addition, shorter injection intervals of the projectiles decrease the asymptotic fouling resistance. Sintering of the fouling layer which hinders the cleaning action of projectiles should be accounted for this phenomenon. Furthermore, all projectiles decreased the induction time of the fouling process. The asymptotic fouling resistance was also approached much quicker compared to the case of no injection. The performance of any projectile lies in a trade-off between its size, texture and stiffness. Stiffness produces a shear force required to dislodge the deposit and size is required to maintain the contact area between projectile and the surface. Accordingly, a criterion was developed to determine the optimum projectile size and stiffness for best cleaning performance. The criterion shows that bigger and softer projectiles cannot last for a long time injection processes. Given the importance of size and stiffness, the projectiles were subsequently divided into two groups of hard and soft due to the required stiffness and velocity to move the projectile within the tube. To discriminate between these two groups, a new term called contact stability factor or Z factor is proposed which is a function of stiffness and size. A mechanistic model has also been developed to predict the asymptotic fouling resistance when projectiles are in operation, based on injection rate, fouling rate and removal rates. The model predicts the asymptotic fouling resistance with an accuracy of 69% based on CaSO4 concentration, saturation concentration, injection interval, shear force and contact stability of the tube with the projectile

Software Evaluation Via a Study of Deviations in Results of Manual and Computer-Based Step-Wise Method Calculations for Shell and Tube Heat Exchangers

Abstract: In this study manual calculation for both shell and tube, sides of a single-phase shell and tube heat exchanger are conducted. The calculations are made using Aspen B-JAC software, results of step-wise method computations are compared with manual calculations. Results show that although more accurate methods like step-wise method include many details in calculations, difference between the yielded results and experimental-based algorithms like Bell-Delaware's method is acceptable

NOTCH3 Expression Is Linked to Breast Cancer Seeding and Distant Metastasis

Background: Development of distant metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which includes dissemination of cancer cells from the primary tumor to secondary organs. NOTCH developmental signaling plays a critical role in promoting epithelial-to-mesenchymal transition, tumor stemness, and metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive. Methods: We have established metastatic xenografts expressing high endogenous levels of NOTCH3 using estrogen receptor alpha-positive (ERα+) MCF-7 breast cancer cells with constitutive active Raf-1/mitogen-associated protein kinase (MAPK) signaling (vMCF-7Raf-1) and MDA-MB-231 triple-negative breast cancer (TNBC) cells. The critical role of NOTCH3 in inducing an invasive phenotype and poor outcome was corroborated in unique TNBC cells resulting from a patient-derived brain metastasis (TNBC-M25) and in publicly available claudin-low breast tumor specimens collected from participants in the Molecular Taxonomy of Breast Cancer International Consortium database. Results: In this study, we identified an association between NOTCH3 expression and development of metastases in ERα+ and TNBC models. ERα+ breast tumor xenografts with a constitutive active Raf-1/MAPK signaling developed spontaneous lung metastases through the clonal expansion of cancer cells expressing a NOTCH3 reprogramming network. Abrogation of NOTCH3 expression significantly reduced the self-renewal and invasive capacity of ex vivo breast cancer cells, restoring a luminal CD44low/CD24high/ERαhigh phenotype. Forced expression of the mitotic Aurora kinase A (AURKA), which promotes breast cancer metastases, failed to restore the invasive capacity of NOTCH3-null cells, demonstrating that NOTCH3 expression is required for an invasive phenotype. Likewise, pharmacologic inhibition of NOTCH signaling also impaired TNBC cell seeding and metastatic growth. Significantly, the role of aberrant NOTCH3 expression in promoting tumor self-renewal, invasiveness, and poor outcome was corroborated in unique TNBC cells from a patient-derived brain metastasis and in publicly available claudin-low breast tumor specimens. Conclusions: These findings demonstrate the key role of NOTCH3 oncogenic signaling in the genesis of breast cancer metastasis and provide a compelling preclinical rationale for the design of novel therapeutic strategies that will selectively target NOTCH3 to halt metastatic seeding and to improve the clinical outcomes of patients with breast cancer

Improving the recovery of "difficult to release" periplasmically-expressed products from recombinant E. Coli

The periplasm of E. coli has been recognized a suitable location for the production of large quantities of many industrially important soluble recombinant proteins, and offers some important advantages over both intracellular and extracellular production. The oxidative environment of the periplasm promotes correct disulphide bonding and protein folding; there is reduced risk of proteolytic attack in the periplasm; and the periplasm accounts for <5% of total cell protein, so that selective release reduces subsequent purification demands. Despite these merits, periplasmic expression systems have not yet fulfilled their true potential, largely due to the lack of reliable general methods for efficient selective release of periplasmically expressed proteins at large-scale. The classical osmotic shock procedure, the only reliable method for releasing proteins from the periplasmic space of E. coli, is expensive and time-consuming, and thus it is not feasible at large scale. The main objective of this study has been to develop a gentle chemical permeabilisation method for selective release of periplasmically-expressed proteins from recombinant E. coli. In the first experiments, the titre, location and form of anti-lysozyme Fab D1.3 were determined during fed-batch cultivation of E. coli. It was shown that the Fab produced as both soluble and insoluble forms and released into the culture medium over the course of fed-batch fermentation. Purification of Fab D1.3 was then performed using various chromatographic methods, and the most effective target Fab purification was achieved by using sequential cation exchange – Protein G affinity chromatography route with an overall yield of 83%. Pure model proteins typically secreted to the periplasm (i.e. beta-lactamase, alpha amylase and Fab D1.3 fragment) were subsequently exposed to various chemicals, and alterations in the secondary structure of the proteins in the presence of various chemicals were investigated by high throughput circular dichroism (ht-CD) system. Chemicals such as 0.1% Triton X-100, 0.05% benzalkonium chloride (BAC), 0.1% cetyltrimethylammonium bromide (CTAB), and 2 M urea remarkably changed the secondary structure of beta-lactamase and alpha-amylase. The secondary structure of Fab D1.3 was more vulnerable to the tested chemicals. The biological activity of the target proteins in the presence of various chemicals was also measured and it was revealed that changes in the secondary structure of proteins do not necessarily cause reduction in the biological activity and vice versa. Concentrations of chemicals which did not reduce the biological activity of the proteins were eventually examined in subsequent periplasmic release experiments using recombinant E. coli strains producing the same target proteins, and the performance of various chemical permeabilisers were evaluated by comparing to classical osmotic shock and mechanical cell disruption. It was demonstrated that low concentrations of chemicals such as sodium deoxycholate (DOC) and/or chelating agents, isoamyl alcohol released the periplasmic proteins as efficient as or more efficient (up to 168%) than osmotic shock treatments. It was also proved that chemicals could increase the periplasmic release efficiency when they used in combinations. For instance, 1 M EDTA in combination with detergents could increase the periplasmic release of beta-lactamase and Fab D1.3 up to 80% and 130%, respectively. Such synergetic effect for release of alpha-amylase and Fab D1.3 was also observed when 1% solvents (hexane, xylene, benzene, toluene, and isoamyl alcohol) were combined with detergents such as 0.025% DOC, 0.01% CTAB and 0.1% Triton X-100

Roles of nucleocapsid proteins and 5' TAR in HIV-1 genomic RNA dimerization

Human Immunodeficiency Virus type 1 (HIV-1) genomic RNA (gRNA) dimerization appears essential for viral infectivity via, among others, facilitating gRNA strand exchange during reverse transcription, but the gRNA dimerization mechanisms remain largely unknown. What is well known about gRNA dimerization is that the dimerization process requires the proteolytic processing of Pr55gag polyprotein into smaller products. If the gag processing is blocked experimentally, it will lead to the formation of low-mobility dimers which is termed immature dimers, as apposed to high-mobility dimers (mature dimers) formed in the wild type HIV-1. Two kinds of dimers have been isolated from HIV-1 particles: Immature dimers isolated from grown-up (≥ 24h old) protease-inactive (PR-in) and from newly released (0-15min old) virions; mature dimers isolated from grown-up wild type and some mutants of HIV-1. My first research project is to identify the roles of NC-containing proteins including Gag polyprotein, NCp15, NCp9, and NCp7 in HIV-1 gRNA dimerization. I showed for the first time that the formation of immature genomic RNA dimers (igRNAds) in protease-inactive (PR-in) HIV-1 is protein-dependent (Gag-dependent, not spontaneous) and the nucleocapsid (NC) portion of the Gag polyprotein chaperones the formation of igRNAds. Based on the effect of 19 mutations studied in grown-up PR-in HIV-1, I showed that the proximal zinc finger and the linker sequences but not the distal zinc finger of the nucleocapsid protein play critical roles in the formation of igRNAds in PR-in HIV-1. However, the basic nature of the basis residues of the N-terminus and the linker are not involved in igRNA dimer formation. Other maturation products of NC, i.e. NCp15, NCp9, and NCp7, have differential roles in HIV-1 gRNA dimerization. Newly released p9 HIV-1 contains much lower level of immature dimers compared to NCp15 and NCp7 HIV-1 and the rate of accumulation of immature dimers is similar to PR-in HIV-1. But, NCp9 is as efficient as NCp7 in the transformation of immature dimers to mature dimers. On the other hand, NCp15 like NCp7 directs the fast accumulation of immature dimers in newly released HIV-1. Nonetheless, NCP15 like Gag polyprotein is quite inefficient in the maturation process, i.e. the transformation of immature dimers to mature dimers. To summarize: NCp7 is efficient in fast formation of immature gRNA dimers and the maturation process; NCp15 is only efficient in the formation of fast immature dimers; NCp9 is efficient in the transformation process but not in the fast formation of immature dimers. The Dimer Initiation Site (DIS) is a dimerization initiation site for all immature gRNA dimers, irrespective of their mechanism of formation. In the second part of my thesis I studied the role of the 5' transactivation response element (TAR) in HIV-1 gRNA dimerization. By making mutations in the upper TAR stem-loop structure including two palindrome sequences, I showed that this region contributes enormously to HIV-1 gRNA dimerization. However, gRNA dimerization was not restored in the compensatory mutants of both palindromes, suggesting that TAR-TAR kissing mechanism is not involved in gRNA dimerization. By jointly mutating TAR and deleting DIS, I showed that the TAR-dependent dimerization is not associated with the DIS. For the first time I showed that the UCU bulge of TAR is very important for HIV-1 gRNA dimerization and we conclude that the role of large TAR mutations in gRNA dimerization is entirely assumed by the TAR bulge.La dimérization de l'ARN génomique (gRNA) du Virus d'Immunodéficience Humaine 1 (VIH-1) est essentiel pour un virus infectieux parce que, entre autres, elle facilite l'échange de brins pendant la transcription inverse. On sait déjà que le processus de dimérisation requiert au moins un certain degré de maturation protéolytique de la polyprotéine Pr55gag, une protéine dont la maturation progressive génère les protéines NCp15, NCp9 et, finalement, NCp7. J'ai d'abord identifié les rôles respectifs de Pr55gag, NCp15, NCp9 et NCp7 dans la dimérisation de l'ARN génomique (ARNg). J'ai montré que la formation de dimères immatures du gRNA (igRNAds) est dépendante de Gag chez les VIH-1 avec une protéase-inactive (PR-in), et que la portion nucleocapside (NC) de Pr55gag est essentielle à cette forme de dimérisation. Me basant sur les effets de 19 mutations étudiées chez les VIH-1 PR-in adultes (vieux de 12 h et plus), j'ai montré que le doigt de zinc proximal et la séquence du linker du NC, mais non son doigt de zinc distal, jouent un rôle critique dans la formation de igRNAds chez les VIH-1 PR-in. Cependant, la nature basique des résidus du N-terminus et du linker ne contribue pas à la formation de igRNAds. Les autres produits de maturation de NC, c.-à-d. NCp15, NCp9 and NCp7, jouent des rôles différentiels dans la dimérisation de l'ARN du VIH-1. Les VIH-1 p9 fraichement sortis contiennent un très faible niveau de dimères immatures par rapport aux VIH-1 p15 et p7, et le taux d'accumulation des ces dimères ressemble à celui qui est observé chez les VIH-1 PR-in. Mais NCp9 est aussi efficace que NCp7 dans la maturation des dimères immatures. Par ailleurs, NCp15 forme des dimères immatures aussi rapidement que NCp7, mais est incapable de les transformer en dimères matures. En résumé: NCp7 est efficace à la fois en formation rapide et en maturation des dimères immatures; NCp15 est efficace uniquement en formation de dimères immatures; NCp9 est efficace en maturation, mais pas en formation de dimères immatures. Le DIS est le site d'initiation de la dimérisation de tous les dimères immatures, peu importe leur mécanisme de formation. Dans la 2ème partie de ma thèse, j'ai étudié le rôle de l'ARN TAR 5' (transactivation response element) dans la dimérisation du VIH-1. En introduisant des mutations dans le haut du TAR tige-boucle, qui contient deux séquences palindromiques, j'ai montré que cette région contribue énormément à la dimérisation de l'ARNg du VIH-1. Cependant, la dimérisation n'a pas été rétablie par des mutations compensatoires dans chacun de ces 2 palindromes, suggérant qu'un mécanisme de TAR-TAR "kissing" n'est pas significatif pendant la dimérisation de l'ARNg. En mutagénisant TAR et en supprimant le DIS, j'ai montré que les mutations dans TAR et dans le DIS avaient des rôles indépendants durant la dimérisation. Pour la première fois, j'ai montré que la boucle UGU de TAR joue un rôle très important dans la dimérisation de l'ARNg, et je conclus que le rôle de TAR dans la dimérisation de l'ARNg est entièrement assumé par la boucle du TAR