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Production and characterization of LEA29Y, a variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin, in Pichia pastoris.
- Source :
-
Applied Microbiology & Biotechnology . Aug2011, Vol. 91 Issue 3, p543-551. 9p. - Publication Year :
- 2011
-
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 = 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 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 01757598
- Volume :
- 91
- Issue :
- 3
- Database :
- Academic Search Index
- Journal :
- Applied Microbiology & Biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 62661587
- Full Text :
- https://doi.org/10.1007/s00253-011-3277-4