Full-length anti-human ferntin antibodies were previously used for successful targeting tumor cells in hepatocellular carcinoma and advanced Hodgkin's disease As a first step toward creation of more effective immunotoxins, the single-chain anti-human ferritin antibody scF11 was expressed in E.coli strain BL21(DE3). The scF11-encoding plasmid contains a fragment of Erwinia carotovora pectate lyase gene pelB encoding the signal peptide, plus the VH domain, linker peptide (GlyiSer)}, and VL domain. The scFn antibody was found m periplasm as insoluble aggregates containing both processed (-21 kDa) and unprocessed (-29 kDa) forms. After solubilizing with 6M GdmHCI, the protein was purified in 5M urea using ion exchange and calcium tartrate gel chromatography. Refolding of the purified scF11 antibody by either rapid dilution or single-step dialysis resulted in only 5-10% of soluble protein In contrast to this, slow dialysis with controlled rate of a decrease in dénaturant concentration provides an 100% yield of soluble active scF11 antibody The scF11 antibody binds to ferritin with association constant (7.8 10" M ') comparable to that of the parent monoclonal F11 antibody (7 0 10s M ') and adopts correct "immunoglobulin fold" as determined by CD and fluorescence spectroscopy. Thermodynamic stability of scF11 was estimated in dénaturant- and pH-induced, and thermal unfolding studies and compared with stability of the parent F11 antibody (mouse lgG2a) measured by scanning calorimetry and spectroscopic probes. (Supported by grants from Byelorussian Soros Foundation, No 96-18-2020-8, Byelorussian Foundation for Basic Research. No 895-211, and Russian Foundation for Basic Research. No 96-04-49548).
|Publication status||Published - 1 Dec 1997|
Martsey, S. P., Kravchuk, Z. I., Chjjrnaneyich, A. A., Vlasov, A. P., Korneva, F., & Deyev, S. M. (1997). Single-chain antibody against human ferritin: Purification, refolding, and stability studies. FASEB Journal, 11(9).