Production and characterization of LEA29Y,a variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin,in <Emphasis Type="Italic">Pichia pastoris</Emphasis> |
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Authors: | Lin Wan Shengyun Zhu Yingying Li Shan Liu Hao Yang Shengfu Li Youping Li Jingqiu Cheng Xiaofeng Lu |
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Institution: | (1) Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China; |
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Abstract: | Blocking the CD28/B7 costimulatory pathway is a promising strategy in the treatment of graft rejection, graft-versus-host
disease and autoimmune diseases. LEA29Y, a high-affinity variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig),
is a more potent inhibitor of the interaction between CD28 and B7 than is CTLA4Ig. In a previous study, LEA29Y was produced
in a mammalian cell system, which is time-consuming and expensive. To obtain LEA29Y more efficiently and cost effectively,
we attempted to produce LEA29Y using a Pichia pastoris expression system. The gene encoding LEA29Y, with an additional 6-His tag at the N-terminus, was cloned into the yeast vector
pPIC9K and expressed in the P. pastoris strain GS115. Under the optimized induction conditions for protein expression (inoculum density, OD600 = 80; methanol concentration added daily, 1.0–3.0%; induction time point, 72–96 h; culture medium pH = 6.0), the yield of
purified LEA29Y was approximately 30 mg l−1 by one-step Ni-agarose affinity chromatography. PNGase F treatment showed the purified LEA29Y to be post-translational modified
by N-linked glycosylation. In biological function assays, LEA29Y expressed in P. pastoris demonstrated specific binding to B7-1/B7-2-positive Raji cells and also suppressed lymphocyte proliferation in a dose-dependent
manner. These results suggest that LEA29Y produced in P. pastoris is biologically active and will be useful for experimental therapy on immunotherapy for transplant rejection and autoimmune
diseases. |
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