ZMARZLY, KATARZYNA,KAROLINA (2016) Harnessing Caenorhabditis elegans secretion to produce recombinant secreted proteins from other nematodes. Masters thesis, Durham University.
|PDF - Accepted Version|
Acanthocheilonema viteae excretory-secretory 62 product can modulate the immune system, and is a potential treatment for rheumatoid arthritis and other autoimmune diseases. It has been shown to inhibit arthritis in mouse and in vitro models. However, the only known way of producing functional ES-62 involves growing A. viteae worms in two hosts (ticks and gerbils), isolating adult worms from infected gerbils and allowing them to secrete ES-62 into liquid media. This method is time-consuming, expensive, difficult to scale up and does not yield sufficient amount of protein for extensive analysis. Standard recombinant expression systems are not sufficient for expressing recombinant ES- 62 in a correctly folded, active form, because they cannot generate the nematode- specific post-translational modifications required for the biological activity of ES-62. The nematode Caenorhabditis elegans provides an alternative expression system. C. elegans is a model organism, with well-understood genetics, that can be easily manipulated, has a fast reproduction rate and can be grown in large cultures. We hypothesised that understanding the biology and sequence of endogenous ES-62, as well as the biology of C. elegans secretion, will help us design the best way to use C. elegans as an alternative expression system for secreted recombinant ES-62.
In this study, we characterised the endogenous ES-62 genomic fragment and found that it had a complex structure with a large second intron. We found that C. elegans secretes a simple mixture of proteins into the media, in a relatively short period of time. The most abundant of those proteins was identified by MALDI-TOF as an aspartyl protease ASP-6. We used control elements of asp-6 to direct expression of ES-62 genomic fragment into the excretory/secretory pathway of C. elegans. Despite strong expression of the transgene, we found that the recombinant protein expression in transgenic C. elegans was weaker than anticipated.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Keywords:||Caenorhabditis elegans, ES-62, secreted parasite proteins, recombinant nematode proteins, Acanthocheilonema viteae, C. elegans excretory-secretory system|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||14 Dec 2016 11:20|