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Production of human fucosyltransferases in Trichoderma reesei

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posted on 29.03.2022, 01:56 by Anna Gryshyna
Glycosylation is the most complex posttranslational modification specific to eukaryotes and also some bacteria. . One of the bottlenecks limiting progress in the glycobiology research is the non-availability and/or high price of glycan-modifying enzymes, such as fucosyltransferases, due to the imperfections of existing production systems. The aim of this project was to evaluate the potential of a filamentous fungus Trichoderma reesei as an expression host for recombinant human ɑ-1,3/4 fucosyltransferases (FUT3 and FUT4). The principles discovered and approaches used can be applied to other mammalian glycoproteins of biomedical interest. In the current work, cDNAs encoding the C-terminal catalytic domains of the human ɑ-1,3/4 fucosyltransferases were synthesised according to the T. reesei codon usage and assembled into expression vectors under the strong cellobiohydrolase 1 (cbh1) promoter. FUT3 and FUT4 were produced as a fusion to a Strep-tag, mCherry fluorescent protein and the core-linker fragment of the endogenous CBH1 protein to facilitate protein purification and secretion. Due to the better performance of the transformants producing the recombinant FUT3 protein, most of the work was carried out with the transformants producing FUT3, while the FUT4 protein was not studied in detail. Screening of T. reesei transformants demonstrated not only secretion of a fusion protein but also its degradation, most probably by the extracellular proteases of T. reesei. Protease inhibition studies suggested that the main protease damaging the recombinant FUT3 was a serine protease active at acidic pH. The recombinant protein was purified using a Strep-Tactin column, and the estimated yield of FUT3 was about 70 mg/l. N-terminal sequencing of purified proteins revealed that some of them were N-terminally blocked; one protease cleavage site was identified at the N-terminus of the mCherry protein. Cultivation of the T. reesei transformants at a neutral pH and with addition of a serine protease inhibitor PMSF demonstrated that degradation of the recombinant FUT3 in the culture supernatants could be suppressed. The activity of the purified recombinant FUT3 was assayed and preliminary results indicated that recombinant FUT3 has certain enzymatic activity although further studies are necessary to confirm these data. In conclusion, it was established that a recombinant human fucosyltransferase FUT3 could be produced in T. reesei. Further studies into optimisation of the cultivation conditions and strain development to limit the proteolytic degradation are required to achieve higher production levels.

History

Table of Contents

1. Introduction -- 2. Materials and methods -- 3. Expression of the recombinant FUT3 and FUT4 in Trichoderma reesei -- 4. Optimisation of culture conditions -- 5. Purification and analyses of purified proteins -- 6. Protease inhibition studies -- 7. Bioreactor cultivations of the CMF3-44 transformant -- 8. Summary, conclusions and future work -- Appendices.

Notes

Theoretical thesis. Bibliography: pages 163-210

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

PhD, Macquarie University, Faculty of Science and Engineering, Department of Chemistry and Biomolecular Sciences

Department, Centre or School

Department of Chemistry and Biomolecular Sciences

Year of Award

2017

Principal Supervisor

Helena Nevalainen

Additional Supervisor 1

Nicolle Packer

Additional Supervisor 2

Liisa Kautto

Rights

Copyright Anna Gryshyna 2017. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (xv, 232 pages)

Former Identifiers

mq:70741 http://hdl.handle.net/1959.14/1267284