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Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
KTH, School of Engineering Sciences (SCI).ORCID iD: 0000-0001-9423-0541
2019 (English)In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 400, no 3, p. 405-415Article in journal (Refereed) Published
Abstract [en]

Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3C(pro)) towards improved proteolytic activity on the substrate LEVLFQ down arrow GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3C(pro) library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.

Place, publisher, year, edition, pages
WALTER DE GRUYTER GMBH , 2019. Vol. 400, no 3, p. 405-415
Keywords [en]
Coxsackievirus B3, FACS, GFP-fusion, intracellular assay, protease engineering, protease substrate profiling
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-245129DOI: 10.1515/hsz-2018-0362ISI: 000458628400014PubMedID: 30521472Scopus ID: 2-s2.0-85058219114OAI: oai:DiVA.org:kth-245129DiVA, id: diva2:1296057
Note

QC 20190313

Available from: 2019-03-13 Created: 2019-03-13 Last updated: 2020-04-29Bibliographically approved
In thesis
1. Amyloid Beta – Biotherapy Target and Biotechnological Tool
Open this publication in new window or tab >>Amyloid Beta – Biotherapy Target and Biotechnological Tool
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease is a progressive neurodegenerative disease characterized primarily by memory impairment and cognitive decline. As of 2020, an estimated 50 million people suffer from Alzheimer’s disease or related dementia and no disease-modifying treatment options are yet approved for clinical practice. A major pathological feature of Alzheimer’s disease is the presence of cerebral senile plaques with aggregated amyloid beta (Aβ) peptides as the main constituent. In this thesis, Aβ is used in five separate studies either as a target for the development of potential biotherapeutical interventions or as a tool in biotechnological research.

In the first study, a high-throughput screening method was developed that enables functiondriven selection of protein-based aggregation inhibitors from combinatorial libraries. The method employs a reporter protein consisting of Aβ42 fused to the N-terminus of green fluorescent protein (GFP). The reporter protein misfolds due to the aggregating nature of Aβ42. Following protein expression in Escherichia coli a low whole-cell GFP fluorescence signal was detected using flow cytometry. However, when co-expressed with an affibodybased aggregation inhibitor, the reporter protein was rescued from aggregation and an increased whole-cell GFP fluorescence signal was detected in flow cytometry. By combining the screening method with flow cytometric cell sorting, the aggregation-inhibiting affibody molecule could successfully be enriched from a large background of non-inhibiting affibody molecules. The results thus demonstrated that the developed method enables highthroughput screening and sorting of combinatorial protein libraries based on the Aβ aggregation inhibiting ability.

The second study explored a strategy to increase the uptake of a biotherapeutical candidate protein into the central nervous system (CNS) via receptor-mediated transcytosis across the blood-brain barrier (BBB). The affibody-based candidate ZSYM73 binds monomeric Aβ and inhibits Aβ aggregation. Here, ZSYM73 was fused to the C-terminus of a single-chain variable fragment (scFv8D3) binding the transferrin receptor (TfR); a receptor expressed on the BBB. An engineered albumin-binding domain (ABD) was fused to ZSYM73 to extend the circulatory half-life of the fusion protein. In a mouse study, the tri-specific fusion protein scFv8D3-ZSYM73-ABD exhibited increased cerebrospinal fluid (CSF) bioavailability compared to the control protein ZSYM73-ABD, indicating an active transport mechanism into the CNS.

In the third study, a novel method for combinatorial protease engineering was developed and applied to generate highly proteolytic active variants of the coxsackievirus 3C protease. The method is based on the findings form the first study and employs a reporter protein consisting of Aβ42 fused to the N-terminus of GFP via a peptide linker containing a protease substrate sequence. The reporter protein misfolds upon expression in E. coli, which resulted in a low whole-cell GFP fluorescence signal detected in flow cytometry. Co-expression of a protease with activity on the substrate sequence led to proteolytic separation of the aggregation-prone Aβ42 peptide from GFP and restored whole-cell fluorescence. This method was used in combination with flow cytometric cell sorting to isolate highly proteolytic active variants from a randomly mutated 3C protease library. The aim of the fourth study was to evaluate the potential of the newly developed method from the third study to engineer the substrate specificity of proteases. A semi-rational tobacco etch virus (TEV) protease library was screened for variants with proteolytic activity on a novel target substrate. The target substrate differed substantially from the wild-type TEV consensus substrate and exhibited high sequence similarity to the aggregation-inducing hydrophobic core region of Aβ. After three rounds of flow cytometric cell sorting, a set of TEV protease variants was enriched that exhibited proteolytic activity on the novel substrate.

In the fifth study, a methodology employing flow cytometric sorting of multiprotein aggregates was developed to investigate the protein interactome related to Aβ plaques. It was demonstrated that in human serum or human CSF, Aβ aggregates bound to a fluorescent probe can be detected and isolated using flow cytometry. Quantitative mass spectrometry analysis was performed on Aβ aggregates isolated from human CSF. The abundances and functional features of proteins found in the isolated aggregates were investigated, and a hypothetical model of the layered architecture of Aβ aggregates was proposed. 

In conclusion, this thesis describes the development of new concepts and methods that will hopefully contribute to increasing the understanding and improving the therapy of Alzheimer’s disease and other diseases associated with protein aggregation. 

Abstract [sv]

Allt liv som vi känner till är till högsta grad beroende av proteiner. Proteiner utför nästan alla biologiska processer inom alla livsdomäner. Proteiner ger struktur till våra celler, transporterar syre i vårt blod, förser oss med energi genom att bryta ner socker och dessutom skyddar de oss från smittsamma sjukdomar, för att bara nämna några exempel. Proteiner består av kedjor av sammanlänkade aminosyror. De flesta proteiner består av ett unikt antal och en specifik ordning av 20 olika aminosyror. Aminosyrakedjorna veckas oftast till tredimensionella proteinstrukturer som gör att de kan utföra sina biologiska funktioner. Följaktligen kan det vara skadligt när veckningsprocessen går fel och proteiner inte antar deras funktionella struktur. Felveckade proteiner associeras med en rad olika sjukdomar. Ett sådant exempel är Alzheimers sjukdom, en irreversibel, progressiv hjärnsjukdom som gradvis leder till försämrat minne och mental förmåga. Psykiska och andra neurologiska symtom är även mycket vanliga och sjukdomen leder ofta till en för tidig död. I hjärnorna hos patienter med Alzheimers sjukdom bildas små klumpar, så kallade senila plack, som består av felveckade och aggregerade proteiner. Huvudbeståndsdelen i dessa klumpar är ett protein som kallas amyloid beta (Aβ).

Protein engineering beskriver processen att generera proteiner med förbättrade eller nya funktioner anpassade efter önskade egenskaper. Idag används såna proteiner inom olika områden, till exempel jordbruk- och livsmedelsindustri, diagnostik- och läkemedelsindustri och biobränsleproduktion. Affinitetsproteiner och proteaser är två typer av proteiner som har varit centrala för studierna i den här avhandlingen. Deras förmåga att specifikt binda till respektive klyva andra målproteiner har lett till en utbredd användning som bland annat forskningsreagens men även som molekyler för terapeutiska ändamål.

I studierna som ligger till grund för den här avhandlingen har amyloid beta dels använts som ett målprotein för att utveckla potentiella terapeutiska proteiner, men även som ett molekylärt verktyg för utveckling av nya metoder för protein engineering, och slutligen för att studera sammansättningen av patologiska proteinaggregat från Alzheimerpatienter.

Den första studien i denna avhandling beskriver utvecklingen av en ny metod för protein engineering för att effektivisera utvecklingen av aggregeringshämmare. Hämningen av Aβaggregering och följaktligen bildning av senila plack är en potentiell mekanism för att behandla Alzheimers sjukdom. I den andra studien konstruerades ett bioterapeutiskt kandidatprotein, som binder till Aβ, för att förbättra upptaget till centrala nervsystemet. Ökat läkemedelsupptag i centrala nervsystemet, platsen där amyloid beta aggregerar, har en stor sannolikhet att öka läkemedlets terapeutiska effekt. I den tredje studien utvecklades en ny metod för att generera proteinklyvande proteaser med förbättrade eller nya funktioner. Denna metod är baserad på aggregering av Aβ-peptiden och genererade ett antal nya proteasvarianter med förbättrad förmåga att klyva ett målprotein. I den fjärde studien användes samma metod för att generera proteasvarianter som klyver ett nytt målprotein som är väldigt likt Aβ. De nya varianterna har potential att vidareutvecklas till proteasbaserade läkemedelskandidater i framtiden. Den femte studien beskriver utvecklingen av en metod för att undersöka sammansättningen av tidigare nämnda senila plack. Den nya kunskapen om sammansättning av plack kan bidra till att förstå sjukdomens patologi, och utveckling av potentiella nya terapier.

Sammanfattningsvis så kommer resultaten som presenteras i denna avhandling förhoppningsvis att bidra till ökad förståelse och förbättrad behandling av Alzheimers sjukdom och andra sjukdomar som associeras med proteinaggregering.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2020
Keywords
Alzheimer’s disease, amyloid beta, blood-brain barrier, green-fluorescent protein, intracellular assays, proteases, protease substrate specificity, protein aggregation, protein engineering.
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-272826 (URN)978-91-7873-507-5 (ISBN)
Public defence
2020-05-29, https://kth-se.zoom.us/webinar/register/WN_QqN3EAF1RvagtaZIodM8Tw, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 2020-05-06

Available from: 2020-05-06 Created: 2020-04-29 Last updated: 2020-05-06Bibliographically approved

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Meister, SebastianHendrikse, NatalieLöfblom, John

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