title: Production and characterization of LEA29Y, a variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin, in Pichia pastoris.
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, OD(600) = 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.
authors.affiliation: Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, People's Republic of China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wan
authors.forename: Shengyun
authors.initials: S
authors.lastname: Zhu
authors.forename: Yingying
authors.initials: Y
authors.lastname: Li
authors.forename: Shan
authors.initials: S
authors.lastname: Liu
authors.forename: Hao
authors.initials: H
authors.lastname: Yang
authors.forename: Shengfu
authors.initials: S
authors.lastname: Li
authors.forename: Youping
authors.initials: Y
authors.lastname: Li
authors.forename: Jingqiu
authors.initials: J
authors.lastname: Cheng
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Lu
mesh_terms: D000069594:Abatacept; D060887:B7 Antigens / Q000378:metabolism*; D018106:CD28 Antigens / Q000378:metabolism*; D060908:CTLA-4 Antigen / Q000276:immunology*; D045744:Cell Line, Tumor; D006801:Humans; D018796:Immunoconjugates / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007136:Immunoglobulins / Q000276:immunology*; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism
chemical_list: D060887:B7 Antigens; D018106:CD28 Antigens; D060908:CTLA-4 Antigen; D018796:Immunoconjugates; D007136:Immunoglobulins; D000069594:Abatacept