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Heterologous expression of human ST6GAL1 in trichoderma reesei

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thesis
posted on 28.03.2022, 10:08 by Aneesh Chandran Bhuvanachandran Pillai
Sialyltransferases can be used to add sialic acid to glycoproteins to study and modify the effects of sialylation on, for example, the metastatic potential of cancer cells, drug resistance, differentiation of stem cells and the half-life and in vivo bioactivity of therapeutic proteins. A functional human sialyltransferase, ST6Gal1, was expressed in the high protein secreting filamentous fungus Trichoderma reesei with a view of improving availability of the enzyme. Two expression cassettes were designed (i) a cassette with the mCherry gene which encodes a fluorescent protein to enable initial screening of transformants as well as tracking of secretion (ST6Gal1 produced as a fusion protein), and (ii) a cassette without mCherry where the ST6Gal1 production was monitored using ST6Gal1 specific antibodies. Both constructs also contained a Strep-tag™ II and an enterokinase cleavage site to facilitate the purification of recombinant ST6Gal1 (rST6Gal1) and to cleave the rST6Gal1 from the fusion protein respectively. A truncated and codon optimised ST6Gal1 cDNA was expressed under the T. reesei cbh1 promoter. Recombinant ST6Gal1 was found to be secreted into the culture medium at 48 h, established by Western blotting with ST6Gal1 specific antibodies. Two main forms, 40 kDa and 60 kDa of the secreted rST6Ga11 were observed indicating processing of the fusion protein. The secreted rST6Gal1 was eventually degraded during cultivation. A total protease activity assay revealed an increase in the extracellular protease activity over a period of 120 h of cultivation. The transformants exhibited a higher protease activity compared to the non-transformant RUT-C30 host. Subsequent analysis of the intracellular fraction by Western blotting revealed that processing of the fusion protein occurred inside the cells. Transcriptional analysis of the ST6Gal1 gene by qRT-PCR revealed that the gene was expressed at 24 h, 48 h and 96 h. Confocal microscopy suggested co-localisation of mCherry in the endoplasmic reticulum (ER) possibly indicating the activity of ER associated quality control mechanism. Further analysis of the RNA levels by qRT-PCR revealed an increase in the transcription levels of genes encoding proteins associated with the unfolded protein response (UPR) and ER associated degradation (ERAD) pathways, such as BiP1, PDI, calnexin, Sec61, ubiquitin and proteasome subunits RPN8 and beta3. This indicated the cellular stress that might have led to the disintegration of rST6Gal1 resulting in lower secretion Southern blot analysis revealed integration of multiple copies of the ST6Gal1 gene into the fungal genomic DNA, with at least one of the transformants having the expression cassette integrated in the cbh1 locus. A lectin based enzyme activity assay was developed and established to be functioning with commercially available recombinant human ST6Gal1. The results were further validated using mass spectrometry. As a summary, this project serves as a proof of concept for the successful expression of a recombinant human sialyltransferase ST6Gal1 in Trichoderma reesei.

History

Table of Contents

Chapter 1. Introduction -- Chapter 2. Material and methods -- Chapter 3. Preparation of expression vectors and generation of trichoderma reesei transformants -- Chapter 4. Expression of human sialyltransferase ST6Gal1 in trichoderma reesei RUT-C30 -- Chapter 5. Degradation of the recombinant ST6Gal1 and determination of functionality of rST6Gal1 produced in trichoderma reesei -- Chapter 6. Summary, future directions and conclusions -- References -- Appendices

Notes

Bibliography: pages 205-251 Empirical thesis.

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

PhD, Macquarie University, Faculty of Science and Engineering, Department of Molecular Sciences

Department, Centre or School

Department of Molecular Sciences

Year of Award

2016

Principal Supervisor

Helena Nevalainen

Additional Supervisor 1

Nicki Packer

Rights

Copyright Aneesh Chandran Bhuvanachandran Pillai 2016. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (xiv, 265 pages) illustrations (some colour)

Former Identifiers

mq:70151 http://hdl.handle.net/1959.14/1260759