Development of a novel fusion system for soluble protein overexpression and purification in Escherichia coli

Recombinant protein production is a useful technology for therapeutic and diagnostic applications. The bacterium Escherichia coli is widely used for the bioproduction of proteins, but still presents some drawbacks. Recombinant proteins with biomedical interest have proved difficult to express properly in this host system, resulting in insoluble protein aggregates. Fusion protein technology has been applied to overcome these drawbacks and to optimize protein expression in E. coli. This research aims at the evaluation of a novel fusion system (Fh8 and H tags) effects on protein production. Target proteins used in this work present biomedical interest and results obtained here showed that these novel fusion tags increased protein production yields. This novel fusion system is a promising tool for the production of recombinant proteins with biomedical interest

Evaluation of a novel Escherichia coli fusion system for the production of recombinant immunogenic proteins

Recombinant protein production has been widely applied for therapeutic and diagnostic applications, namely for polyclonal antibody production. Antibodies are usually raised against a specific protein by immunisation of animals with the purified protein. The Escherichia coli host cell is widely used for the bioproduction of proteins with biomedical interest but some of them are still difficult to express properly in this host system, resulting in insoluble protein aggregates. Gene fusion technology has been employed to optimise recombinant protein production in E. coli. Fusion partners have also been used to increase protein immunogenicity. In this work, the immunopotentiating properties of a novel fusion partner (H partner) were studied. The H partner was fused to three target proteins with diagnostic interest: CP12, a 12 kDa surface protein from Cryptosporidium parvum oocysts; CWP, a cyst wall protein from Giardia lamblia; and ENT, a surface protein from Entamoeba histolytica trophozoites. The results obtained here show the H partner as a promising tool for immunodiagnostic and immunoprophylactic purposes

Development of a novel fusion system for recombinant protein production and purification in Escherichia coli

Tese de doutoramento Programa Doutoral em Engenharia Química e BiológicaProteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it presents several issues limiting the efficiency of this production. The aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting problem found when expressing eukaryotic proteins in E. coli. Gene fusion technology has been widely used for the improvement of soluble protein expression and/or purification in E. coli. Fusion partners/tags are highly soluble and stable proteins that promote target protein solubility, possibly because of the protection of nascent polypeptides from the cytoplasm milieu and the given conditions for their proper folding. The Fh8 is a calcium-binding protein from excreted-secreted antigens of Fasciola hepatica that showed to be highly soluble and stable when produced as a recombinant protein in E. coli. This recombinant protein is also a useful tool for the diagnosis of fasciolosis, and its N-terminal sequence of eleven amino acid residues (denominated as H) was suggested to play an important key role in the production and immunological properties of the entire protein. These Fh8 interesting features suggested this protein usage as a fusion partner for enhanced recombinant protein production in E. coli. This is the main focus of this thesis work. The Fh8 and H peptides were analyzed as expression and solubility fusion partners, and at the same time, compared to commonly used fusion tags (Chapter 2). A broad range evaluation was conducted using six target proteins, eight fusion tags, two different induction conditions, and four E. coli expression strains. The results showed that Fh8 acts as an effective solubility enhancer tag, being ranked among the best solubility tags. It is also an excellent candidate to be used with other fusion tags in parallel high throughput screenings, presenting advantages over large tags for the evaluation of protein solubility. Results from this work also showed that the H tag did not function as a solubility enhancer tag, but it improves protein expression levels in E. coli. The H tag was then suggested for the recombinant production and adjuvant-free administration of immunogens (Chapter 3). By using a 12-kDa antigen from Cryptosporidium parvum as example, the H tag demonstrated to be an attractive tool for the production of polyclonal antibodies, overcoming several limitations of the process, namely, the availability of antigen, its immunogenicity, adverse effects of adjuvants, and unspecific antibody production. The Fh8 fusion partner was further investigated as purification handle (Chapter 4). Taking into account its calcium-binding properties, the Fh8 offered a rapid, inexpensive and efficient singlestep purification of biologically active target proteins via hydrophobic interaction chromatography (HIC), and under mild conditions. The efficiency of this purification strategy was comparable to that obtained using the His6 tag, and both purification technologies were also combined into a dual affinity strategy to improve further the purity level of target proteins. Proteins purified by the Fh8-HIC strategy have the extra feature of being free of E. coli endotoxins. Taking into account the versatility of the Fh8 fusion system, a novel strategy for the soluble production of bone morphogenetic protein-2 (BMP-2) and interleukin-10 was developed (Chapter 5). Fh8 fusion proteins were directly soluble produced in E. coli, presenting dimeric and oligomeric ordered conformations. The Fh8BMP-2 was, however, not functional, rising intriguing questions about the final structure of the fusion protein. Indeed, the Fh8 might direct the BMP-2 to a soluble folding pathway, but BMP-2 presents a different conformation from that required for its biological function. Two novel variants of Fh8 were then developed as fusion partners, conducting the mutation of its single cysteine residue to alanine or tyrosine (Chapter 6). Fh8Ala and Fh8Tyr fusion proteins achieved similar solubility as Fh8-fused ones, presenting less calcium-dependent conformational changes and less oligomer forms than the Fh8-fused ones. These two mutations demonstrated the importance of the cysteine residue in Fh8 oligomerization, though other residues may also contribute to this state. This thesis work reported for the first time the efficient use of the calcium-binding protein Fh8 as a promising gene fusion technology. The Fh8 fusion system is a robust tool for the recombinant protein production in E. coli, combining four main skills into such a small partner: protein expression, solubility, purification and immunogenicity.As proteínas são atualmente produzidas em diversas fábricas celulares microbianas, sendo a Escherichia coli o hospedeiro dominante na produção de proteínas recombinantes. Contudo, este hospedeiro apresenta alguns problemas que limitam a eficiência desta produção, sendo a agregação em corpos de inclusão insolúveis uma das suas principais limitações na expressão de proteínas eucariotas em E. coli. A tecnologia de fusão genética tem sido muito usada para melhorar a expressão e purificação de proteínas solúveis em E. coli. Os tags /parceiros de fusão são proteínas altamente solúveis e estáveis que promovem a solubilidade de proteínas alvo, possivelmente conferindo-lhes proteção no citoplasma, reunindo as condições necessários para o seu folding adequado. O Fh8 é uma proteína de ligação ao cálcio, excretada-secretada pelos antigénios do Fascíola hepatica, que mostrou ser altamente solúvel e estável quando produzida em E. coli. O Fh8 é também uma ferramenta útil no diagnóstico da fasciolose, e a sua sequência N-terminal de onze aminoácidos (denominada de H) poderá ser importante na produção e nas propriedades imunológicas da proteína total. Todas estas características do Fh8 realçam o seu possível uso como parceiro de fusão para uma melhor produção de proteínas recombinantes em E. coli, sendo esse o objetivo principal do presente trabalho. Os péptidos Fh8 e H foram analisados como parceiros de expressão e solubilidade, tendo sido também comparados com outros parceiros de fusão regularmente usados (Capítulo 2). Realizouse uma profunda avaliação com recurso a seis proteínas alvo, oito tags de fusão, duas condições de indução diferentes e quatro estirpes de expressão de E. coli. Os resultados mostraram que o Fh8 é de facto um parceiro de fusão que promove a solubilidade, enquadrando-se entre os melhores tags de solubilidade. O Fh8 é também um bom candidato para ser usado com outros tags em extensas avaliações paralelas, apresentando vantagens em relação a tags maiores na avaliação da solubilidade das proteínas. Os resultados deste trabalho também mostraram que o tag H não funcionou como parceiro de solubilidade, mas este parceiro de fusão aumentou os níveis de expressão de proteínas em E. coli. O tag H foi usado para a produção recombinante de imunogénios e para a sua administração sem adjuvantes (Capítulo 3). Usando um antigénio de 12 kDa de Cryptosporidium parvum como exemplo, o tag H demonstrou ser uma ferramenta interessante para a produção de anticorpos policlonais, contornando várias limitações do processo, tais como, a disponibilidade do antigénio, a sua imunogenicidade, os efeitos adversos dos adjuvantes e uma produção de anticorpos pouco específica. O parceiro de fusão Fh8 foi também estudado como tag de purificação (Capítulo 4). Tendo em conta as suas propriedades de ligação ao cálcio, o Fh8 possibilitou uma purificação rápida, barata e eficiente de proteínas biologicamente ativas numa só etapa, através da cromatografia de interação hidrofóbica (HIC) e em condições moderadas. Esta estratégia de purificação teve uma eficiência semelhante à obtida usando o tag His6, e estas duas tecnologias de purificação foram ainda combinadas numa dupla purificação para aumentar o nível de pureza das proteínas alvo. A purificação por Fh8-HIC apresenta a mais-valia de obter proteínas livres de endotoxinas de E. coli. Tendo em conta a versatilidade do sistema de fusão Fh8, desenvolveu-se uma nova estratégia para a produção solúvel da bone morphogenetic protein-2 (BMP-2) e da interleukin-10 (Capítulo 5). As proteínas de fusão com o Fh8 foram diretamente produzidas de forma solúvel em E. coli, apresentando dímeros e oligómeros numa conformação ordenada. No entanto, a Fh8BMP-2 não foi funcional, levantando questões sobre a estrutura final da proteína de fusão. O Fh8 poderá direcionar a BMP-2 para uma via de folding solúvel, sendo que, no entanto, a BMP-2 apresenta uma conformação diferente daquela exigida para a sua atividade biológica. Neste trabalho, foram ainda desenvolvidos dois novos parceiros de fusão variantes do Fh8, tendo-se efetuado uma mutação no único aminoácido cisteína para alanina ou tirosina (Capítulo 6). As proteínas de fusão com Fh8Ala e Fh8Tyr alcançaram uma solubilidade semelhante à obtida pelas proteínas de fusão com Fh8, apresentando uma menor alteração conformacional dependente de cálcio e uma menor oligomerização comparativamente às proteínas de fusão com Fh8. Estes dois mutantes demonstraram a importância do aminoácido cisteína na oligomerização do Fh8; outros aminoácidos podem, porém, contribuir para este estado. Este trabalho apresentou pela primeira vez o uso eficiente da proteína de ligação ao cálcio, o Fh8, como uma tecnologia de fusão genética promissora. O sistema de fusão Fh8 é uma ferramenta robusta para a produção de proteínas recombinantes em E. coli, combinando quatro características principais num pequeno tag: expressão proteica, solubilidade, purificação e imunogenicidade

The novel Fh8 fusion technology for protein expression in Escherichia coli: a comparison with the traditionally used fusion systems

[Excerpt] The recombinant expression of natural proteins in Escherichia coli is limited by the lack of efficient methods for soluble production. Several efforts have been made to overcome this limitation, including the genetic fusion of highly soluble protein domains (fusion partners) to the target proteins. Among the available fusion partners, the E. coli maltose-binding protein (MBP), glutathione S-transferase (GST) and N-utilization substance A (NusA) are often used to enhance protein solubility. However, due to their large size, these partners can be problematic for structural and functional analyses, requiring their removal using specific proteases. The removal of the fusion partner is not always successful and the resulting cleaved target protein may also precipitate into insoluble aggregates.info:eu-repo/semantics/publishedVersio

A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli : Its application to a 12 kDa antigen from Cryptosporidium parvum

The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimise recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP 12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunisation. The H tag (of only 1 kDa) efficiently triggered a CP 12-specific immune response, and it also improved the immunisation procedure without requiring coadministration of adjuvants. Moreover, polyclonal antibodies raised against the HCP 12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP 12 antigen.The financial support of Fundacao para a Ciencia e Tecnologia (FCT), Portugal, is acknowledged: Project PTDC/CVT/103081/2008 (co-financed by COMPETE) and grant SFRH/BD/46482/2008 (POPH-QREN) to Costa SJ. We would like to thank Lurdes Delgado and Sonia Soares for the Cryptosporidium oocysts isolation from fecal samples, and also to Hitag (R) Biotechnology, Ltd for kindly providing the H and Fh8 tag sequences used in this work

The Fh8 tag : a fusion partner for simple and cost-effective protein purification in Escherichia coli

Downstream processing is still a major bottleneck in recombinant protein production representing most of its costs. Hence, there is a continuing demand of novel and cost-effective purification processes aiming at the recovery of pure and active target protein. In this work, a novel purification methodology is presented, using the Fh8 solubility enhancer tag as fusion handle. The binding properties of Fh8 tag to a hydrophobic matrix were first studied via hydrophobic interaction chromatography (HIC). The Fh8 tag was then evaluated as a purification handle by its fusion to green fluorescent protein and superoxide dismutase. The purification efficiency of the Fh8-HIC strategy was compared to the immobilized metal ion affinity chromatography (IMAC) using the His6 tag. Results showed that the Fh8-HIC binding mechanism is calcium-dependent in a low salt medium, making the purification process highly selective. Both target proteins were biologically active, even when fused to Fh8, and were successfully purified by HIC, achieving efficiencies identical to those of IMAC. Thus, the Fh8 acts as an effective affinity tag that, together with its previously reported solubility enhancer capability, allows for the design of inexpensive and successful recombinant protein production processes in Escherichia coli.This work was conducted with the financial support of the Fundacao para a Ciencia e Tecnologia (FCT), Portugal, by the fellowship SFRH/BD/46482/2008 (POPH-QREN) to Sofia J. Costa. The FCT project PTDC/CVT/103081/2008 (co-funded by COMPETE) and QREN co-promotion project number 3515 are also acknowledged. The authors thank Dr. Huseyin Besir, Protein Expression and Purification Facility Core, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, for supplying the pETM11 expression vector, and also Dr. Vitor Costa for kindly providing the SOD target gene used in this work