Tissue factor is a transmembrane glycoprotein that functions as the primary initiator
of coagulation in response to mechanical or chemical damage. Due to its key
position within the coagulation cascade it also plays an important role in the
pathology of thrombosis and thrombotic complications associated with
cardiovascular disease as well as in non-thrombotic disorders and diseases such as
obesity, diabetes, cancer and HIV-AIDS. Recognising the potential in tissue factor
inhibition as a novel approach to antithrombotic therapy, our laboratory utilized
phage display technology in a previous study, in order to identify a 26 kDa single
chain antibody fragment which functionally inhibits human tissue factor.
In the current study, the tissue factor inhibitory single chain variable fragment (TFIscFv)
was expressed by means of the pIT2 plasmid vector by Escherichia coli
HB2151. This expression system was utilised in an up-scale setting in an attempt to
improve the TFI-scFv yield. Although functional TFI-scFv was successfully purified
from the culture media by means of Protein A affinity chromatography, the process
was hampered by large sample volumes, low levels of expression as well as the high
cost involved in Protein A purification.
Due to an initial focus on improving TFI-scFv yield through the processing of larger
sample volumes rather than the improvement of the expression system, immobilised
nickel affinity chromatography was investigated as a more cost effective alternative
to Protein A affinity chromatography. It was found that the original expression system
was incompatible with immobilised nickel affinity chromatography as the protein was
expressed into the culture media. The culture media contained nickel chelating
elements that stripped the nickel from the column and consequently prevented TFIscFv
purification.
Subsequently the TFI-scFv gene was isolated, cloned into an over-expression
system and modified to redirect the expression to the bacterial cytoplasm. Although
TFI-scFv was successfully redirected to the bacterial cytoplasm and purified by
means of nickel affinity chromatography, it was found that expression was hampered
due to the presence of rare codons. The detrimental effect of rare codons on TFI scFv yield was addressed through the modification of the expression host by the coexpression
of the pRARE plasmid as well as by the rare codon optimization of the
TFI-scFv gene sequence for expression in E. coli. Although the co-expression of the
pRARE plasmid only slightly improved TFI-scFv yield, a sufficient amount of TFIscFv
was generated for functional testing. The modified TFI-scFv displayed a similar
inhibition effect with reference to the original construct. The rare codon optimisation
resulted in a substantial increase in TFI-scFv yield but consequently resulted in the
loss of solubility and the production of inclusion bodies. Although the loss of TFIscFv
solubility is unwanted, the high level of expression achieved provides an ideal
platform for the further development and characterization TFI-scFv in animal
thrombosis models.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-08232012-145143 |
| Date | 23 August 2012 |
| Creators | Vermeulen, Jan-G |
| Contributors | Prof E van Heerden, Prof SM Meiring |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-08232012-145143/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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