A Novel Strategy For Approaching MG Therapy By Using "Target-directed Attack"

Acetylcholine receptor (AChR) is a kind of ligand-gated ion channel transmembrane protein, consisting of five subunits: 2α, β, γ (ε), δ. Among them, a subunit is the main immunogen of autoimmune disease Myasthenia gravis (MG). Abnormal anti-AChR antibodies (AChRAb) existing in MG patients can bind to AChR α67-76, α 125-147, α 183-200, reducing functional AChR amounts and inhibiting AChR binding ability with ACh, in turn hampering the neuromuscular transmission. Therefore, the investigation of pathogenesis and therapy for MG has been focused on the main functional region of AChR a- subunit. The main contents of this present work will be described as follows.Firstly, the open reading frame of AChR_α205 was cloned into various vectors containing different promoters, such as pUC118, pET32M and pMAL-c2 and expressed in E. coli BL21. However, the target protein can be expressed as soluble form only with recombinant strain E. coli BL21/ pMAL-AChR_α205, the expression amount of fusion protein MBP- AChR_α205 was about 30% of total proteins in cells. This is the first report for the soluble expression of AChR_α205 in prokaryotic cell. The fusion protein was efficiently purified by amylose affinity column and also could be cleaved to be a single AChR_α205 by protease Factor Xa. Furthermore, the affinity column, amylose-MBP-AChR_α205 can also be used as an immunoadsorbent. It was shown from immunoadsorption test that about 75% - 98% AChRAb can be specifically removed from anti-AChR mice sera, whilst serum protein and IgG, IgM, IgA still maintain the normal level. In addition, more than 90% AChRAb in two MG patient sera were eliminated with this immunoadsorbent, the removal rate is higher than those chemical immunoadsorbents, compared with reported references. It was also demonstrated that neither MBP-AChR_α205 or AChR_α205 was leaked out during the immunoadsorption,as detected by ELISA. (Li et al, Biotechnol Lett, 2004; Guo et al, J Immunol Methods, 2005)Secondly, the target gene AChR^os was extended to AChRaiii and then inserted into different exprssion vectors such as pVT, pGAPZaA and pPIC9K to form recombinant plasmid. After transformation and expression, the target protein AChRa2n was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K- AChRasn with a yield of 25 mg/L. Then, the target protein was purified by Q-Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-activated Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two MG patient sera reached 84% and 94%, respectively. (Guo et al, Zhonghua Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 2005)Thirdly, since the mechanism of protein self-splicing was elucidated, many recombinant proteins have been rapidly and efficiently purified by intein self—splicing reaction. However, the application of intein-mediated protein ligation (IPL) in protein engineering is still un-matured. In this work, a protocol of this technology was performed and some results were obtained. A fusion protein, CySH-gelonin-intein-CBD was expressed in the form of inclusion bodies in E. coli BL21. After denaturation and renaturation with step-wise dialysis, about 60% fusion protein can be refolded. The refolding protein can be bound to chitin beads by affinity adsorption and the single gelonin can be cleaved and eluted from chitin beads after incubation at pH 6.5, 25 °C for 48 h. The result indicated that the recovery of CySH-gelonin reached about 1.2 mg/L and was also active, as tested by cell-free protein synthesis in vitro. On the other hand, AChR^n-intein-CBD was also expressed in recombinant cell with the same procedure as described above. After the fusion protein was renatured and cleaved with 100 mM MESNA, AChRa2ii bearing COSR at its C terminus was formed based on the mechanism of protein self-splicing. Afterwards, the N-terminal CySH of...