Development of an insect cell factory for the production of complex biopharmaceuticals using a synthetic biology approach

Tese de mestrado. Biologia (Biologia Celular e Biotecnologia). Universidade de Lisboa, Faculdade de Ciências, 2011Insect cells, in particular the Spodoptera frugiperda Sf9 cell line, are a popular system for the production of biologically active recombinant proteins. However, the current technology uses baculovirus infection which has two main disadvantages: firstly, the recombinant gene is only expressed transiently during the infection cycle, after which cells die; secondly, due to the lytic nature of this system, the cellular protein processing machinery is severely compromised at the end of the infection cycle, affecting the correct formation of recombinant proteins whose expression is usually controlled by very-late baculovirus promoters. Stably transformed insect cell lines represent an alternative system for continuous protein production. However, their establishment is laborious, requiring the identification of cell clones that display the right expression properties due to random integration of the gene-of-interest. Furthermore, independently of the expression system, multimeric biopharmaceuticals continue to have very low yields, mainly because the stoichiometry between components is not properly reached in the producer cells. To overcome these issues, this report shows work towards the development of a novel Sf9 cell factory combining (i) recombinase mediated cassette exchange (RMCE) for targeted gene integration and (ii) a tetracycline (Tet) inducible system for the modulation of gene expression levels. We started by evaluating the strength of different promoters to drive GFP expression in Sf9 cells as well as different transfection protocols. The baculovirus promoter OpIE2 allowed the strongest expression when compared to others, and the efficiency of transfection was significantly higher using the lipotransfection method. From the different transfection protocols, 4 cell populations with distinct fluorescence distributions were obtained after 3 weeks under hygromycin selection. These populations were independently subject to limiting dilution and isolated cell clones were analyzed by southern blot to screen for single-copy integration of the tagging GFP construct. While the majority of clones were single copy, cassette exchange was performed in a few clones by co-transfection with flippase and the target plasmid containing dsRed. Cells were selected in the presence of neomycin and cassette exchange was confirmed by PCR of genomic DNA, revealing the absence of tagging cassette and the presence of the target cassette in the some locus. In parallel, the functionality of a tetracycline inducible system in Sf9 cells was evaluated by substituting mammalian promoters with insect cell specific promoters. However, transactivation was suboptiomal, requiring additional changes to the original system. In summary, this work reports for the first time the implementation of a RMCE system in Sf9 cells and a preliminary assessment of Tet transactivation in these cells. This new cell line will be a major breakthrough since it will combine the advantages inherent to Sf9 cell growth, continuous protein production, the ability of re-using a well characterized locus for targeted DNA integration and inducible gene expression.A cultura de células de insecto conjugada com a infecção por baculovirus representa um dos sistemas biológicos de eleição para a produção de proteínas recombinantes biologicamente activas. Há vários biofármacos em ensaios clínicos e alguns já no mercado, como é o caso da vacina contra o vírus do papiloma humano (Cervarix, GSK), que utilizam este tipo tipo de sistema de expressão. No entanto, o uso de baculovirus representa um sistema de expressão transiente / lítico inerente ao processo de infecção. Em contraste, quando estavelmente transformadas, as células de insecto podem ser usadas como um sistema de expressão contínuo e não-lítico. Contudo, o desenvolvimento de linhas celulares estáveis é significativamente demorado e laborioso, sendo necessário identificar e isolar clones que exibam elevadas taxas de expressão. A grande variabilidade existente nos níveis de expressão entre clones deve-se ao chamado efeito de posição, ou seja a aleatoriedade de integração no genoma da célula. Além disso, e independentemente do sistema de expressão, quando se pretende expressar biofármacos multiméricos, como as particulas semelhantes a vírus, estes continuam a ter rendimentos muito baixos. A estequiometria entre os componentes é um factor chave e, quando não é alcançada adequadamente nas células produtoras, resulta na formação de uma grande percentagem de partículas incorrectamente formadas. De forma a superar estas limitações, neste trabalho é iniciado o desenvolvimento de uma linha celular derivada de Spodoptera frugiperda, usando uma combinação de duas tecnologias: (i) sistema de troca de cassete mediada por recombinase (RMCE) e (ii) circuito transcricional indutível. Os sistemas RMCE utilizam um eficiente e avançado processo de troca génica localizada por intermédio de uma reacção de recombinação. Anteriormente implementada em células de mamífero, este tipo de tecnologia foi agora aplicada num cenário de expressão estável em células de insecto com o objectivo de permitir o uso repetido de locais genómicos pré-caracterizados com elevada taxa de expressão. Já os circuitos indutíveis sintéticos, como o sistema de indução por tetraciclina, permitem ajustar a expressão de diferentes genes de interesse por intermédio de um indutor, facilitando a produção de produtos multiméricos correctamente formados. O trabalho desenvolvido nesta dissertação de mestrado começou por centrar-se na análise de diferentes promotores no controlo da expressão de genes repórter na linha celular Sf9. Foram estudados promotores com diferentes origens mas com uma base de funcionamento comum em células de insecto, como os promotores de baculovirus OpIE2 e OpIE1, o promotor da metalotioneína e o das proteínas de choque térmico (hsp70) de Drosophila. Em resultado, verificou-se que o promotor OpIE2 superou consideravelmente os restantes na expressão de eGFP. Por outro lado, o promotor da metalotioneína induzido por cobre revelou ser um sistema desadequado para expressão de proteínas de interesse em células Sf9, uma vez que apenas para doses citotóxicas se verificou uma expressão de eGFP mensurável. Para o funcionamento do sistema RMCE são necessários dois vectores distintos e complementares (Tagging / Target). Em ambos os vectores foi utilizado o promotor OpIE2 para a expressão das proteínas repórter (diferentes nos dois vectores: Tagging – dsRed; Target - eGFP), e o promotor OpIE1foi usado para a expressão do agente de selecção, necessário para garantir a expressão estável dos dois vectores no genoma da célula (Tagging – higromicina; Target -neomicina). Enquanto a integração do plasmídeo Tagging no genoma se dá através de um processo aleatório, a integração do Target é o resultado da troca bem-sucedida por recombinação enzimática. A selecção pelo agente neomicina assegura o sucesso da troca génica uma vez que o gene de resistência é activado pela inserção de um códão de iniciação (ATG) presente apenas no Target. Antes do processo de troca, é importante que apenas uma cópia da cassete Tagging seja integrada no genoma da célula para que o sistema RMCE seja funcional. Utilizaram-se dois métodos em paralelo para transfectar as células Sf9 com o plasmídeo Tagging: lipotransfecção usando cellfectin e eletroporação, donde resultaram quatro populações celulares com distribuições de fluorescência distintas. Para cada população de células foi posteriormente realizada uma diluição progressiva para a obtenção de clones produtores, os quais foram analisados por citometria de fluxo para comparar o nível de expressão de eGFP. Posteriomente, foi implementado um protocolo de Southern blot para analisar o número de cópias integradas da cassete Tagging em cada clone., Dos nove clones analisados, apenas um revelou ter mais do que uma cópia. Em alguns dos clones com apenas uma cópia procedeu-se à co-transfecção da construção Target com a enzima recombinase (flippase). Foi possível seleccionar células resistentes à neomicina sugerindo o sucesso da troca de cassetes, tendo sido mais tarde confirmado por meio de PCR do DNA genómico e de RT-PCR do RNA dos genes repórteres.No que toca o circuito indutível de transcrição, o trabalho conduzido teve como objectivo fazer uma avaliação preliminar da sua funcionalidade em células Sf9 antes da implementação directa na cassete de recombinação. No entanto, os resultados revelaram uma fraca indução por parte do agente tetraciclina, justificando a necessidade de uma optimização do sistema original. Em conclusão, com este trabalho reportamos pela primeira vez a implementação bem sucedida de um sistema de troca de cassete por intermédio de recombinação enzimática em células Sf9. Esta tecnologia representa um grande avanço na produção de novas linhas celulares de insecto, combinando as vantagens inerentes à cultura deste tipo de células com a possibilidade de atingir elevados níveis de expressão contínua de diferentes proteínas recombinantes através da fácil integração de genes de interesse no genoma celular

The O-antigen flippase Wzk can substitute for MurJ in peptidoglycan synthesis in Helicobacter pylori and Escherichia coli

The peptidoglycan (PG) cell wall is an essential component of the cell envelope of most bacteria. Biogenesis of PG involves a lipid-linked disaccharide-pentapeptide intermediate called lipid II, which must be translocated across the cytoplasmic membrane after it is synthesized in the inner leaflet of this bilayer. Accordingly, it has been demonstrated that MurJ, the proposed lipid II flippase in Escherichia coli, is required for PG biogenesis, and thereby viability. In contrast, MurJ is not essential in Bacillus subtilis because this bacterium produces AmJ, an unrelated protein that is functionally redundant with MurJ. In this study, we investigated why MurJ is not essential in the prominent gastric pathogen, Helicobacter pylori. We found that in this bacterium, Wzk, the ABC (ATP-binding cassette) transporter that flips the lipid-linked O- or Lewis- antigen precursors across the inner membrane, is redundant with MurJ for cell viability. Heterologous expression of wzk in E. coli also suppresses the lethality caused by the loss of murJ. Furthermore, we show that this cross-species complementation is abolished when Wzk is inactivated by mutations that target a domain predicted to be required for ATPase activity. Our results suggest that Wzk can flip lipid II, implying that Wzk is the flippase with the most relaxed specificity for lipid-linked saccharides ever identified

Evolved to Break Free: Evolution and Diversity in Single Gene Lysis

mall lytic phages compelled by the same evolutionary pressures as large dsDNA phages have evolved mechanisms to lyse their hosts efficiently and at optimal times to ensure maximum fecundity. The ssDNA Microviridae and ssRNA Leviviridae, with extremely small genomes (<6kb), effect lysis with a single gene. The most studied single-gene lysis systems (SGL) are gene E of the canonical microvirus PhiX174 and genes A2 and L of the paradigm ssRNA phages Qβ and MS2, respectively. All three lysis proteins lack muralytic activity, and two, A2 and E, have been shown to function as “protein antibiotics” by acting as noncompetitive inhibitors of conserved peptidoglycan (PG) biosynthesis enzymes, MurA and MraY, respectively. The third, L of MS2, does not inhibit PG biosynthesis but instead is hypothesized to trigger host autolytic response through an unknown mechanism. The work described in this dissertation identifies a chaperone involved in MS2 L-mediated lysis, the minimal lytic domain of L, and a new paradigm in SGL that resolves a long-standing debate in peptidoglycan biosynthesis. Using a genetic approach, dnaJ was shown to be required for L-mediated lysis. DnaJ was shown to form a complex with L and this interaction is abrogated in the absence of the highly basic N-terminal half of L. Mutational analysis of L has revealed that most critical residues are clustered around a highly-conserved LS motif. To determine if SGL evolved at different genomic locations target other steps in PG biosynthesis we studied of the lysis mechanism of Lys^M from an E. coli phage M. Both genetic and biochemical analysis have demonstrated that Lys^M targets MurJ, the proposed Lipid II flippase candidate, and blocks Lipid II flipping to effect host lysis. Thus, the results presented in this dissertation provide the most convincing evidence in favor of MurJ being the Lipid II flippase. Additionally, the results highlight the plasticity of ssRNA phages to evolve inhibitors against proteins involved in PG biosynthesis and maintenance

Structural and mechanistic investigation of a proton-dependent lipid transporter involved in lipoteichoic acids biosynthesis

Staphylococcus aureus (S.aureus) is a successful opportunistic human pathogen, causing superficial infections such as skin and soft tissue infections as well as invasive fatal ones including endocarditis, pneumonia and septicemia with high mortality. It represents one of the growing public health concerns worldwide since the rapid spreading of multi-antibiotic resistant S.aureus strains has increased the failure of therapeutics, urgently calling for pathogenesis research and development of effective treatment strategies. 30% of the population carries S.aureus on the skin and mucous membranes where the pH is usually mild acidic (average 4 to 6). Managing survival under this environment is a critical step for S.aureus successful colonization, dissemination and infection. The cell wall, a multi-layered protective structure, plays a crucial role in maintaining S.aureus viability under hostile surroundings. Lipoteichoic acids (LTA) are one type of the main components of the S.aureus cell wall, composed of repeating glycerol phosphate units and a glycolipid anchor called diglucosyl-diacylglycerol (Glc2-DAG) that fixes LTA polymers on the outer leaflet of the plasma membrane. Glc2-DAG is synthesized in the cytoplasm and transferred across the plasma membrane by the integral membrane protein LtaA (lipoteichoic acid protein A). The deletion of LtaA in S.aureus led to the alteration of the LTA anchor from Glc2-DAG to diacylglycerol (DAG) and attenuated virulence during animal infection. LtaA belongs to transporters that mediate glycolipid translocation. This category is closely involved in the cell wall biosynthesis by transferring multiple precursors or molecules to satisfy the proper assembly of the cell wall. Bacteria harbor diverse glycolipid transporters, most of which are not well understood. LtaA was predicted to belong to the major facilitator superfamily (MFS), a large family of membrane proteins that are ubiquitously distributed in all kingdoms of life, transferring a broad range of substrates from sugars, peptides to ions and lipids across membranes. Research on MFS transporters involved in glycolipids transport is scarce, hindering the understanding of their flipping mechanisms. In this study, we determined the structure of LtaA by X-ray crystallography at a resolution of 3.3 Å. LtaA presents the canonical MFS fold with 12 transmembrane helices (TMs) arranged in two pseudo-symmetric sub-domains, N-domain (TM1-6) and C-domain (TM7-12). A striking feature is the presence of a large amphiphilic central cavity which we hypothesized to accommodate the amphiphilic substrate Glc2-DAG. By analyzing LtaA crystal structure, along with site-direct mutagenesis and transport assays in vitro, we demonstrated that the di-glucosyl moiety of Glc2-DAG is recognized by multiple conserved hydrophilic residues located in the N-terminal domain and loaded to the central cavity. We also proposed a proton-coupling mechanism where E32 undergoes protonation/deprotonation, and this extra driving force allows Glc2-DAG to be translocated at a higher rate. By investigating LtaA function in S.aureus, we revealed that the proton-coupling mechanism allows LtaA to act as an environmental pH sensor and contribute to the survival of S.aureus under an acidic environment. Our results provided insights into the molecular basis of Glc2-DAG flipping and made LtaA a novel target for the development of anti-S.aureus therapeutics

Generating Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) gene deletions in Bladder Cancer Cell Lines

Bladder cancer was responsible for 200,000 deaths worldwide in 2018, it has a well-documented ability to evade and resist chemotherapy treatment. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes are a family of enzymes and form part of the innate immune system in human cells where they mutate cDNA from infecting viruses. 2 APOBECs: APOBEC3A (A3A) and APOBEC3B (A3B) have been linked not only to bladder cancer, but to half of all cancers. These APOBECs leave a distinct mutational signature which has led to many computational studies into APOBEC involvement in cancers. APOBECs seem to have a direct role in evading chemotheraputic agents; through upregulation in cancers and increased mutational signature after rounds of chemotherapy. Because of this it has been postulated that development of techniques to downregulate APOBEC expression in cancers, alongside traditional chemotherapy drugs, will stop recurrence. To test this theory cancer-cell lines with homozygous knockouts for A3A and/or A3B must be created to assess the link between these genes and resistance to traditional chemotherapy drugs. This thesis attempts to create these knockouts in BFTC-905, T24 and 5637 bladder cancer cell lines. A3A was successfully knocked out in one line: BFTC-A2-F, and A3B was potentially knocked out in BFTC-Both3-F and BFTC-Both4-F. This experiment has laid the foundation for more A3A and A3B knockouts to be cloned from pools of knock-out targeted cell lines: 5637, T24 and BFTC-905 or through BFTC-A2-F which can be used as a platform to attempt to knock out A3B too. It would appear that trying to knock-out both A3A and A3B at the same time creates some unexpected results, possibly due to how close these genes are together (~30kb) in the APOBEC3 locus. Deletion of A3B appears hard to achieve, as only a heterozygous deletion in BFTC-B5-F was achieved, these results echo a previous experiment in a normal cell line

An inducible Cre mouse line to sparsely target nervous system cells, including Remak Schwann cells

Nerves of the peripheral nervous system contain two classes of Schwann cells: myelinating Schwann cells that ensheath large caliber axons and generate the myelin sheath, and Remak Schwann cells that surround smaller axons and do not myelinate. While tools exist for genetic targeting of Schwann cell precursors and myelinating Schwann cells, such reagents have been challenging to generate specifically for the Remak population, in part because many of the genes that mark this population in maturity are also robustly expressed in Schwann cell precursors. To circumvent this challenge, we utilized BAC transgenesis to generate a mouse line expressing a tamoxifen-inducible Cre under the control of a Remak-expressed gene promoter (Egr1). However, as Egr1 is also an activity dependent gene expressed by some neurons, we flanked this Cre by flippase (Flpe) recognition sites, and coinjected a BAC expressing Flpe under control of a pan-neuronal Snap25 promoter to excise the Cre transgene from these neuronal cells. Genotyping and inheritance demonstrate that the two BACs co-integrated into a single locus, facilitating maintenance of the line. Anatomical studies following a cross to a reporter line show sparse tamoxifen-dependent recombination in Remak Schwann cells within the mature sciatic nerve. However, depletion of neuronal Cre activity by Flpe is partial, with some neurons and astrocytes also showing evidence of Cre reporter activity in the central nervous system. Thus, this mouse line will be useful in mosaic loss-of-function studies, lineage tracing studies following injury, live cell imaging studies, or other experiments benefiting from sparse labeling

Direct Visualization of Individual mRNA Export Through Differential Fluorescent Labeling

Department of Biomedical EngineeringThe nuclear export of mRNA through the nuclear pore complex is a crucial step in gene expression and is tightly regulated through signal-recognizing proteins. Though the mRNAs in the cytoplasm are well imaged in fixed or living cells, the detection of the export event has been difficult due to the lack of adequate technique to distinguish nuclear and cytoplasmic mRNAs, while it is crucial in elucidating the mechanism of controlling their life cycle. We developed a novel fluorescent labeling tool that will mark the nuclear and cytoplasmic mRNAs differently. Using the tool, we can potentially trace the full life cycle of mRNAs from transcription, splicing, to translation. We present our preliminary results on developing the labeling tool and tracking the live mRNA export dynamics.ope

E. coli Group 1 Capsular Polysaccharide Exportation Nanomachinary as a Plausible Antivirulence Targetin the Perspective of Emerging Antimicrobial Resistance

Bacteria evolving resistance against the action of multiple drugs and its ability to disseminate the multidrug resistance trait(s) across various strains of the same bacteria or different bacterial species impose serious threat to public health. Evolution of such multidrug resistance is due to the fact that, most of the antibiotics target bacterial survival mechanisms which exert selective pressure on the bacteria and aids them to escape from the action of antibiotics. Nonetheless, targeting bacterial virulence strategies such as bacterial surface associated polysaccharides biosynthesis and their surface accumulation mechanisms may be an attractive strategy, as they impose less selective pressure on the bacteria. Capsular polysaccharide (CPS) or K-antigen that is located on the bacterial surface armors bacteria from host immune response. Thus, unencapsulating bacteria would be a good strategy for drug design, besides CPS itself being a good vaccine target, by interfering with CPS biosynthesis and surface assembly pathway. Gram-negative Escherichia coli uses Wzy-polymerase dependent (Groups 1 and 4) and ATP dependent (Groups 1 and 3) pathways for CPS production. Considering E. coli as a case in point, this review explains the structure and functional roles of proteins involved in Group 1 Wzy dependent CPS biosynthesis, surface expression and anchorage in relevance to drug and vaccine developments