Emberson, L.M. and Trivett, A.J. and Blower, P.J. and Nicholls, P.J. (2005) Expression of an anti-CD33 single-chain antibody by Pichia pastoris. Journal of Immunological Methods, 305 (2). pp. 135-151. ISSN 0022-1759 .
CD33 is a cell surface glycoprotein expressed on cells of myelomonocytic lineage, leukaemic cells, but not haematopoietic stem cells. By virtue of its expression pattern, CD33 has become a popular target for new immunotherapeutic approaches to treat acute myeloid leukaemia. The methylotrophic yeast Pichia pastoris strain KM71H was used to produce an anti-CD33 single chain variable fragment (scFv), with the intention of conjugation to a radioisotope, for therapeutic use. To direct secreted expression of the anti-CD33-scFv the alpha-mating factor secretory signal sequence (alpha-MF) was used, with constructs containing a complete (CS) and incomplete (INCS) cleavage site to accommodate the potential outcomes of dibasic endopeptidase, Kex2, and dipeptidyl amino peptidase, Ste13, processing. The anti-CD33-scFv was expressed in BMMY cultures using both constructs, with a final yield of 48 mg/l (CS) and 11 mg/l (INCS). N-terminal sequencing showed that the CS-scFv had not been cleaved by Ste13, leaving amino acids EAEA at the N-terminus. The INCS-scFv construct produced a mixture of 50% authentic scFv and 50% with 11 amino acids from the alpha-MF remaining at the N-terminus. Despite the aberrations in alpha-MF processing, the anti-CD33-scFv's produced from both constructs were found to be functional. Flow cytometry and Biacore analysis demonstrated binding to target antigen CD33 on the surface of human leukaemic cell line HL-60, and to recombinant soluble CD33 respectively.
|Additional information:||0022-1759 (Print) Journal Article Research Support, Non-U.S. Gov't|
|Uncontrolled keywords:||Antigens, CD/*immunology Antigens, Differentiation, Myelomonocytic/*immunology Binding Sites, Antibody/immunology Cloning, Molecular Flow Cytometry HL-60 Cells Humans Immunoglobulin Fragments/*biosynthesis/genetics/immunology Immunoglobulin Variable Region/*biosynthesis/genetics/immunology Immunotherapy/methods Jurkat Cells Leukemia, Myeloid, Acute/immunology/therapy Pichia/genetics/*immunology Recombinant Fusion Proteins/biosynthesis/genetics/immunology Sequence Analysis, Protein Surface Plasmon Resonance Transformation, Genetic|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||Sue Davies|
|Date Deposited:||05 Sep 2008 23:01|
|Last Modified:||05 Sep 2011 23:56|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/7160 (The current URI for this page, for reference purposes)|
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