Kotha, Arun Kumar (2011) The characterisation of a recombinant cytosolic shuttle based on ricin toxin. PhD thesis, University of Greenwich.

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Abstract

Cytosolic access is rate limiting for potential therapeutics such as antisense agents. In nature, several entities deliver macromolecules to the cytosol, one example being ricin toxin (RT). In this thesis, a cytosolic drug delivery system based upon attenuated RT was characterised. The toxicological consequences of mutating RT A chain (RTAC) amino acids: Glu177, Ala178, Ala179, Arg180, Phe181, Gln182 to Gly (mut) or deleting (del) them entirely, was evaluated. Epitopes (i.e., 6His, HA) were also incorporated onto either the C- or N-terminus of the RTAC. The resultant plasmid constructs were sequenced, mapped and expressed in E.coli BL21*DE3. A plasmid encoding RT B chain (RTBC) was also characterised, mapped and expressed in E.coli BL21*DE3. Recombinant proteins were affinity purified from bacterial lysate and were soluble. Proteins were detected at approximately the predicted molecular weight using commercial antibodies specific for RTAC, RTBC or the epitopes incorporated into the constructs (i.e. 6His or V5). Optimal culture conditions were obtained by monitoring protein expression in 1.5mL bacterial cultures. Expression levels were established using Western blotting and immuno-detection (using a 6His specific primary antibody). The optimal culture conditions for RTAC with a C-terminal 6His and HA epitope (clone 175) and RTAC with amino acids 177-182 mutated to Gly (and also containing a C-terminal 6His, HA and Lys, Asp, Glu, Leu (KDEL) motif) (clone 189), were found to be 2h pre-induction incubation, induction (with 0.25mM IPTG) and a 4h post-induction incubation, cultured at 37oC. Upon further characterisation, recombinant RTBC (rRTBC) was found not to be lectinic, unlike commercial RTBC (cRTBC). The toxicity of RTAC mutants was characterised in vitro (B16 and Vero cells) using the MTT assay. Using the B16 cell model, at concentrations up to10g/mL, no IC50 values could be established for rRTAC (clone 189). The published wild type RTAC IC50 was 1.4g/mL in B16 cells. The aforementioned rRTAC (clone 189) was re-associated with cRTBC to form a preparation containing enriched heterodimers. Heterodimeric rRTAC (clone 189) and cRTBC did not show any cytotoxicity in B16 cells at RTAC concentrations of up to 1g/mL. The toxicity of heterodimeric rRTAC (clone 189) and cRTBC was measured in Vero cells and the IC50 (0.39g/mL), although statistically different (P<0.001) from cRTBC associated with cRTAC (at concentrations between 0.01 and 100 ng/mL) may be attributed to cRTBC. It was critical to know if the reduced toxicity of the rRTAC (clone 189) - cRTBC, constructs relative to RT holotoxin, was attributable to the RTAC being unable to access the cytosol. Sub-cellular fractionation was performed using Vero cells and the majority of the immuno-reactive rRTAC (clone 189) was detected in a crude cytosolic fraction after 4h. Having ascertained that the mutations introduced into RTAC limit the toxicity of this molecule, the RT based drug delivery system was compared with a previously well-characterised polymer based drug delivery system. In order to make this comparison, a recombinant cargo protein was required. As gelonin (Gel) had been previously used as a cargo for poly(amidoamine) (PAA) mediated cytosolic delivery, three recombinant gelonin analogues were produced. The first was designated mature gelonin (mGel), the second was pre-gelonin with an additional C-terminal cysteine residue (Gel+Cys) and the third contained pre-gelonin sequence with no additional cysteine residue (Gel-Cys). The Gel-Cys construct was necessary to control for any toxicity limiting effects being exerted by additional C-terminal sequence (found on pre-gelonin). The three gelonin constructs were successfully cloned, sequenced and expressed in E.coli BL21*DE3. All of the recombinant gelonin constructs were detected using commercial antibodies (anti-6His and V5). The cytotoxicity of enriched recombinant gelonin proteins was compared with commercial gelonin (cGel). All of the gelonin preparations were non-toxic up to a concentration of 10μg/mL in B16 cells. Gelonin was then used to evaluate two things: 1) the efficiency of PAA mediated delivery relative to cRTBC and 2) the effect of the inclusion of dithiopyridine monomers upon PAA mediated Gel delivery. The PAA FF103 mediated an increase in cytotoxicity when applied to B16 cells in conjunction with a sub-toxic concentration of either with cGel or rGel (i.e. at a Gel concentration of 1.4μg/mL). This indicated that Gel was delivered into the cytosol by PAA FF103. At 10μg/mL of polymer, 93.75% cell viability was recorded and this was reduced to 16% when 1.4μg/mL cGel was added to an equivalent concentration of PAA FF103 (a statistically significant difference (P<0.001)). An investigation into the trafficking of the FITC-conjugated PAAs, ISA1-FITC and ISA23-FITC, supported PAA mediated cytosolic delivery. In this experiment, ISA1-FITC and ISA23-FITC polymers were non-toxic up to a concentration of 3mg/mL in both Vero and B16 cells and the accumulation of ISA1-FITC in the nucleus was observed after 5h. No increase in cGel toxicity was documented after it was mixed with cRTBC relative to cGel delivery mediated by PAAs. Further, despite being able to ablate RTAC toxicity, cRTBC toxicity remains a problem. Lack of lectinic activity of rRTBC (clone 204) suggests that this is unlikely to be resolved, which limits the application of this delivery system.

Item Type:	Thesis (PhD)
Additional Information:	uk.bl.ethos.571422
Uncontrolled Keywords:	cytoplasm, cell biology, ricin toxin,
Subjects:	Q Science > QH Natural history > QH301 Biology
Pre-2014 Departments:	School of Science School of Science > Department of Pharmaceutical, Chemical & Environmental Sciences
Last Modified:	22 Mar 2017 14:01
URI:	http://gala.gre.ac.uk/id/eprint/8762

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