Expression And Purification Of Soluble ScFv1956#

Myasthenia gravis (MG) is an autoimmune disease caused by anti-AChR antibodies and involved in activation of complement. It is known that scFv of MIR specificity, being univalent, does not cross-link the AChR and thus does not cause antigenic modulation. Also, lacking of Fc, it does not activate the complement system causing disruption of the postsynaptic membrane. These scFv are capable of protecting the AChR against loss induced by the auto-antibodies against AChR,and are potentially useful in the treatment of MG.Previous attempts to express soluble scFv have been made by fusion a secretion signal peptide pelB to the N terminal of the scFv. Soluble antibody fragments are harvested from the peroplasm of E.coli or from the culture supernatant after induction with IPTG. However, the disadvantages of this strategy are low yield and high expenses for purification, which hamper the preparation of soluble scFv in large quantities. In this research, the single chain antibody scFv1956# was expressed in E.coli using a strategy of fusing this fragment to the NusA tag in the hope of obtaining large quantity of soluble product.I subcloned the DNA fragment that encodes the structural gene of anti-AChR scFv1956# from vector pHEN1 into the prokaryotic expression vector pET44a(+) which could add a NusA tag to the N-terminal of the target protein. The reconstructed vector pET44a(+)-scFv1956# is then transformed into the expression host BL21 (DE3) pLysS and Roseta-garmi B(DE3); at the same time, the original vector pHEN1-scFv1956# is transformed into the expression host HB2151 and JM109. In the subsequent experiments, two parallel sets of induction conditions with different temperature and induction time were attempted.The quantity of soluble product under different vectors,host strain and induction conditions were compared by SDS-PAGE and by Western Blot.The PCR product was a 785 bp fragment as predicted. Sequencing analysis confirmed the cloned scFv1956# sequence was correctly inseted into the pET44a(+) vector. The SDS-PAGE and Western blot results indicated that the expression of soluble product using the reconstructed vector pET44a-scFv1956# was higher than the original vector pHEN1-scFv1956#. The yield of soluble antibody fragments from the peroplasm of E.coli or the culture supernatant can not be improved by changing the host strain or induction conditions, with pHEN1-scFv1956#. However, with vector pET44a-scFv1956#, an obvious increase in the soluble product was observed when the cultures were inducted at lower temperature and prolonged induction time. This trend seems most significant with expression host strain BL21(DE3)pLysS. Using Ni2+ affinity chromatography, the soluble His-tagged fusion protein was isolated and then the NusA tag was removed by Thrombin digestion. Western Blot conformed that the soluble product with a relative molecular weight of 27 kD bear c-MYCtag.In conclusion, fusing the NusA tag can help scFv1956# to fold correctly, so as to increase soluble product. When the cultures were inducted at lower temperature and prolonged induction time, the soluble product significantly increased with vector pET44a-scFv1956#. Although the fusion protein needs further purification and thrombin digestion for removing the NusA tag, this strategy was proved of significance in large scale preparation of scFv1956# and in the treatment of MG at molecular level.