Background:
Interleukin-1 receptor accessory protein (IL-1RAP) is one of the
most promising therapeutic targets proposed for myeloid leukemia.
Antibodies (Abs) specific to IL-1RAP could be valuable tools for
targeted therapy of this lethal malignancy. This study is about the
preparation of a difficult-to-produce single-chain variable fragment
(scFv) construct against the membrane-bound isoform of human IL-1RAP
using Escherichia coli (E. coli).
Methods:
Different approaches were examined for refolding and
characterization of the scFv. Binding activities of antibody fragments
were comparatively evaluated using cell-based enzyme-linked
immunosorbent assay (ELISA). Homogeneity and secondary structure of
selected scFv preparation were analyzed using analytical size exclusion
chromatography (SEC) and circular dichroism (CD) spectroscopy,
respectively. The activity of the selected preparation was evaluated
after long-term storage, repeated freeze-thaw cycles, or following
incubation with normal and leukemic serum.
Results:
Strategies for soluble expression of the scFv failed. Even with
the help of Trx, ≥ 98% of proteins were expressed as inclusion bodies
(IBs). Among three different refolding methods, the highest recovery
rate was obtained from the dilution method (11.2%). Trx-tag
substantially enhanced the expression level (18%, considering the
molecular weight (MW) differences), recovery rate (˃1.6-fold), and
binding activity (˃2.6-fold increase in absorbance450nm). The produced scFv exhibited expected secondary structure as well as acceptable bio-functionality, homogeneity, and stability.
Conclusion:
We were able to produce 21 mg/L culture functional and stable
anti-IL-1RAP scFv via recovering IBs by pulse dilution procedure. The
produced scFv as a useful targeting agent could be used in scheming new
therapeutics or diagnostics for myeloid malignancies.