Recombinant expression and enzymatic characterisation of Trypanosoma vivax cathepsin L-like protease (TviCATL) for single chain variable fragment antibody production.
Loading...
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Humans and animals in sub-Saharan Africa are at risk of African trypanosomiasis (AT),
caused by tsetse fly-transmitted protozoan blood parasites of the Trypanosoma genus. Animal
African trypanosomiasis (AAT), or nagana, is caused by T. brucei, T. congolense and T. vivax
and negatively impacts livestock farming and consequently the economy of the continent.
Since AAT occurs in rural areas, affordable rapid diagnostic tests (RDTs) and drugs are
required. Diagnostic tests focus on antibody detection; however, antigen detection is more
favorable since anti-trypanosome antibodies persist in blood for years following recovery. Due
to the parasite’s defense by antigenic variation, development of a vaccine is unlikely.
Molecules that are essential for parasite survival, such as peptidases, are currently being
targeted for diagnosis and chemotherapy. A cathepsin-L-like cysteine protease from T. vivax,
TviCATL, is released by dying parasites in the host bloodstream and was shown to be a
diagnostic target for detecting host antibodies. To achieve diagnosis of current infections,
detection of TviCATL is being explored. The overall aim of this study was to enzymatically
characterise TviCATL; and to study the interaction of antibodies against the TviCATL antigen
which could be used as a chemotherapeutic drug for the diagnosis of T. vivax infections. The
protease, TviCATL, was recombinantly expressed in E. coli using the pET-28a expression
vector and purified using a nickel chelate affinity column. The resulting 47 kDa protein was
identified using western blot and was shown to hydrolyse H-D-Ala-Leu-Lys-AMC and was
inhibited by bestatin and E-64 and had optimal activity between pH 6.5 and 7.5. The crossreactivity
between TviCATL and antibodies produced against other Trypanosoma spp cysteine
proteases was evaluated in western blots, and results confirmed cross-reactivity. In addition,
chicken anti-TviCATL antibodies were able to detect TviCATL in TviCATL-spiked bovine
serum. The production of antibodies using the Nkuku® phage library was employed as an
alternative to the animal-based antibody production and single-chain variable fragment
(scFvs) antibodies were selected by panning against the TviCATL antigen. After four rounds
of panning, TviCATL-scFvs binders were enriched and four clones gave the highest signal
when evaluated using a monospecific ELISA. Due to the low values obtained, optimisation of
panning is necessary for improved results. Optimisation of recombinant expression and
purification of the identified scFvs for use in a sandwich ELISA were explored to this end. This
study showed that TviCATL is a promising chemotherapeutic and diagnostic target for African
animal trypanosomiasis.
Description
Masters Degree. University of KwaZulu-Natal, Pietermaritzburg.