Jorge João

Class 2019

PhD Grant:  PD/BD/150335/2019

PhD thesis

Biomanufacturing of Non-Viral Protein Nanocages for Biotechnological and Biomedical Applications

Supervisor

• Miguel F. Prazeres, Professor @ IST/ULisboa, PT

Proposal summary

Non-viral protein nanocages are highly ordered nanometer scale architectures, which are typically formed by homo- or hetero-self-assembly of multiple monomers into symmetric structures with different dimensions and morphologies. The intrinsic characteristics of protein nanocages make them very attractive as a biological nanomaterial and include a high surface/volume ratio, multi-functionality, ease to modify or manipulate genetically or chemically, high stability, monodispersibility and biocompatibility. Several applications have been implemented in a wide range of scientific areas such as biotechnology, biomedicine, bioimaging, biomineralization and nanomaterial synthesis. In particular, the biotechnological and biomedical applications have been extensively investigated, showing that protein nanocages can be a promising and an interesting tool. The development of this type of applications for non-viral protein nanocages requires large amounts of pure and well-folded assemblies. Consequently, the availability of more efficient biomanufacturing processes is needed to transform protein nanocages into valuable products. Moreover, these processes should be supported by analytical techniques adequate for the quality control and characterization of the biological function and structure of the obtained nanocages. In this context, the main objective of this PhD Project is the development of scalable and cost-effective processes for the biomanufacturing of non-viral protein nanocages. Two models of non-viral protein nanocages, one natural, the small heat shock protein (sHSP) from Methanococcus jannaschi (MjsHSP nanocages), and the other artificial, the trp RNA-binding attenuation protein (TRAP) nanocages, are used throughout the work. The initial step consists in the cloning of the genes encoding the MjsHSP and the TRAP into expression vectors appropriate for in vivo production using Escherichia coli and Vibrio natriegens expression systems. Next, regarding the upstream processing, the production of both model nanocages in E. coli cells is set-up and used as a benchmark manufacturing method. Additionally, the same production is performed in V. natriegens cells, followed by an optimization process of several conditions considered more relevant according to the literature. Afterwards, regarding the downstream processing, a purification strategy is also developed using the non-viral protein nanocages both produce in E. coli and V. natriegens. The process is implement based on chromatographic processes, being optimized in different parameters and conditions in order to obtain a highly purified product. The biological structure of the protein nanocages synthesized in vivo in both bacteria is studied and characterized in detail using a range of conventional and alternative analytical techniques. Characterization of the protein nanocages include in vitro internalization cell assays to verify the biological activity.

PUBLICATIONS

Papers

João J, Lampreia J, Prazeres DMF, Azevedo AM, Manufacturing of bacteriophages for therapeutic applications, Biotechnology Advances, 49:107758, 2021, doi:10.1016/j.biotechadv.2021.107758

Oral Presentations

João J, Rosa SS, Azevedo AM, Prazeres DMF, Chromatographic purification of small heat shock protein nanocages, 40th ISPPP – International Symposium on the Separation of Proteins, Peptides and Nucleotides, November 7-10, 2021, Porto, Portugal

Poster Presentations

João J, Prazeres DMF, Biomanufacturing of protein nanocages for biomedical applications, PhD Open Days 2022, November 14-16, 2022, Instituto Superior Técnico, Lisbon, Portugal

João J, Rosa SS, Azevedo AM, Prazeres DMF, Purification of small heat shock protein nanocages for biomedical applications, 2nd iBB – Institute for Bioengineering and Biosciences Workshop, September 23, 2022, Instituto Superior Técnico, Lisbon, Portugal

João J, Rosa SS, Azevedo AM, Prazeres DMF, Development of a chromatographic approach for the purification of small heat shock protein nanocages, 3rd BioIberoAmerica – Ibero-American Congress on Biotechnology, April 7-9, 2022, Braga, Portugal

João J, Rosa SS, Azevedo AM, Prazeres DMF, A chromatographic strategy for the purification of small heat shock protein nanocages, MICROBIOTEC’21 – Congress of Microbiology and Biotechnology 2021, November 23-26, 2021, virtual mode

João J, Prazeres DMF, Purification of small heat shock protein nanocages through a chromatographic approach, PhD Open Days 2021, November 22-24, 2021, Instituto Superior Técnico, Lisbon, Portugal

João J, Rosa SS, Azevedo AM, Prazeres DMF, Chromatographic purification of small heat shock protein nanocages, 15th SBCN – International Symposium on Biochromatography and Nanoseparations, May 17-20, 2021, virtual mode (recognized with the Poster Prize – VIJI Awards SBCN 2021)

DOCTORAL PROGRAM (36 ECTS)

Curricular Units:

• General Doctoral Training (6 ECTS)

• Advanced Experimental Techniques and methodologies (6 ECTS)

• Bioentrepreneurship (6 ECTS)

• Advanced Topics in Bioengineering and Biosciences (6 ECTS)

• Nanobiotechnology (6 ECTS)

• Outreach and Teaching Skills – Department of Bioengineering (6 ECTS)

Advanced courses:

• Workshop “LaTeX 101 – A soft introduction to scientific typesetting”, organized within the PhD Open Days 2022 – Instituto Superior Técnico, November 16, 2022 (3 hours)

• Workshop “Writing of the PhD Thesis and preparation of the document”, organized within the PhD Open Days 2022 – Instituto Superior Técnico, November 16, 2022 (1,5 hours)

• Course “Introduction to Python and Machine Learning for the Biosciences – 4th Edition”, organized in virtual mode by the Instituto de Investigação e Inovação em Saúde (i3S) – University of Porto, October 17-21, 2022 (20 hours)

• Event “ACS on Campus – University of Lisbon”, organized by the American Chemical Society (ACS) at Instituto Superior Técnico, August 26, 2022, Lisbon, Portugal (4 hours)

• Course “Scientific Tools in Biological Assays: Principles of Cell Cultures and Genetics”, organized in virtual mode by the Faculty of Pharmacy – University of Lisbon, March 17-19, 2022 (16.5 hours)

• SPICA Virtual Session “The Importance of Chromatography in the Development of Vaccines”, organized in virtual mode, October 14, 2021 (2 hours)

• Workshop “Escrita Científica – Da observação da realidade à publicação de um artigo: um processo e um resultado”, organized in virtual mode by the Library of Instituto Superior Técnico, June 15-24, 2021 (6 hours)

• Técnico Career Workshop “How to build a CV by BCG”, organized in virtual mode by the Technology Transfer Office – Instituto Superior Técnico, April 20, 2021 (2 hours)

• Técnico Career Workshop “LinkedIn (Básico) by Deloitte”, organized in virtual mode by the Technology Transfer Office – Instituto Superior Técnico, April 5, 2021 (2 hours)

• Workshop “How to Prepare an Excellent Presentation (or at Least an Acceptable One)?”, organized in virtual mode within the PhD Open Days 2020 – Instituto Superior Técnico, October 28, 2020 (3 hours)

• Cell Culture-based Viral Vaccines Course, 5th edition, organized in virtual mode by the European Society of Animal Cell Technology (ESACT), September 28 – October 2, 2020 (25 hours)

• 17th International PhD Student Symposium “Horizons in Molecular Biology”, organized in virtual mode, September 14-17, 2020 (13 hours)

• Champalimaud Research Symposium 2019 – Tissue Environment in Health and Disease, sponsored by the International Union of Biochemistry and Molecular Biology (IUBMB), October 8-10, 2019, Lisbon, Portugal (18 hours)

• Workshop “Teaching STEM Students – Transformation Guide for Teaching Assistants, 3rd Edition”, organized by the Academic Development Office – Instituto Superior Técnico, September 18-19, 2019, Lisbon, Portugal (6 hours)