Successful production of soluble and active single domain antibody against human IL-1RAP

Successful production of soluble and active single domain antibody against human IL-1RAP
Aref Farokhi-Fard,¹ Fatemeh Davami,^2,*
1. Biotechnology research center, Pasteur institute of Iran, Tehran, Iran
2. Biotechnology research center, Pasteur institute of Iran, Tehran, Iran
Introduction: Acute myeloid leukemia (AML) is the most prevalent leukemia in adults with the lowest survival rate. Targeted therapy could be a valuable strategy against AML. Antibodies are of most examined targeting agents. Interleukin 1 receptor accessory protein (IL-1RAP) is an excellent biomarker for targeted therapy of myeloid leukemia. Although the presence of partner domain is necessary for human and murine VH domain to be stabilized, single domain antibodies (sdAbs) as the tiniest antigen binding immunoglobulins could be designed by engineering of VH domain. The production of active and stable VH domain in sufficient quantity is practically problematic due to folding difficulty and aggregation propensity of un-engineered VH. So the reports are extremely rare in this area. Here, we report the bacterial production of a stable un-engineered VH domain against human IL-1RAP. This study may be beneficial for further analyses of single domain antibody properties.
Methods: The coding sequence of anti-IL-1RAP scFv were extracted from a patent (PCTlUS20131077323: SEQ ID NO 13 for VH and SEQ ID NO 14 for VL) which is about a murine antibody developed against 12 residues peptide that is exclusively present in membrane anchored isoform of human IL-1RAP. The scFv coding DNA fragment (VH-(G4S)5-VL arrangement) was artificially synthetized and sub-cloned in pET32a expression vector at NcoI and XhoI sites. Colonies were screened by colony PCR using T7 universal primers. Plasmid extracted from PCR+ clone (C13) was confirmed by PCR and enzymatic digestion. C13 plasmid was then transformed to T7 promoter-based expression host, Origami B strain and the transformants were selected on LB agar plate containing kanamycin, tetracycline and ampicillin. A single colony of Origami B/C13 was cultured in Luria-Bertani (LB) broth medium with the same antibiotics, induced (0.05 mM IPTG, 30 °C, 200 rpm for overnight) and subjected to purification by Ni-NTA affinity resin (Qiagen) in native condition. The eluted protein was concentrated and buffer exchanged to 50 mM Tris-HCL pH 8 by use of centrifugal amicon filter (10 KDa cut off). Protein quantification was accomplished by NanoDrop (Abs280nm, at extinction coefficient of 43,890 M-1 cm-1). Binding activity was evaluated by cell based enzyme-linked immunosorbent assay (ELISA) duplicately on K-562 cells fixed by formaldehyde with different concentrations of Trx-VH and using polyclonal anti-His-tag antibody.
Results: The recombinant protein was successfully purified by NiNTA. The productivity (calculated after concentration and buffer exchange) was estimated to be round 5 mg/L LB culture. The eluted protein exhibited an apparent molecular weight (MW) of ~ 30 kDa (15 kDa lighter than the expected MW). So, we speculated that the lost fragment was pertaining to VL, because the scFv had no C-terminal his-tag and could only be purified through the His-tag located on the upstream of VH. Analysis of insoluble pellet of the centrifuged lysate by SDS PAGE showed a very intense band of 45 kDa. So, we came to the conclusion that the truncation is due to severe proteolysis which removed the VL domain. Binding assessment of purified protein showed that produced VH domain was active, however, as expected, the affinity was relatively low (OD450nm values of 0.05, 0.185 and 0.205 at concentrations of 25, 50 and 100 µg/ml of Trx-VH respectively).
Conclusion: We fortuitously get substantial quantities of soluble active VH domain from a scFv coding construct. We guessed that the proteolysis may be related to the Trx, because, we previously expressed and purified this scFv construct (without Trx) in soluble form in cytoplasm of SHuffle strain. This assumption, however need more researches. Despite the general agreement on instability of un-engineered VH domain, the VH domain we produced was completely stable even after concentrating to 3 mg/ml. This could be explained by in vitro solubility enhancement effect of Trx-tag. We believed that overall avidity derived from several VH in targeted drug delivery vehicles could trivialize the low affinity of single VH domain. More investigations are needed for detailed insight into structural properties and binding kinetics of the described antibody fragment.
Keywords: AML, single domain antibody, scFv, IL-1RAP