ScienceAsia 23 (1997): 159-170 |doi: 10.2306/scienceasia1513-1874.1997.23.159

EXPRESSION OF A RECOMBINANT SINGLE CHAIN Fv ANTIBODY FRAGMENT IN MAMMALIAN CELLS

WILAIWAN CHOTIGEAT^a AND STEPHEN M MAHLER^b

ABSTRACT: Expression of recombinant antibodies and antibody fragments in procaryotic production systems is characterized by low yields and sometimes a reduced affinity for antigen. These problems have been attributed in part to folding constraints in procaryotes, and may be overcome by expressing in eucaryotic production systems. This work is an attempt to produce scFvs of WM65 in mammalian expression system. Since the scFvs of WM65 recognizes a cell surface antigen on normalleukocytes and leukemic cells therefore it CQuld have potential therapeutic application for treatment of chronic lymphocytic leukemia (Ca) and for T-cell depletion of bone marrow to prevent the graft rejection in bone marrow transplantation. The cDNA for scFvs of WM65 in the pHEN1 bacterial expression vector was amplified and modified using polymerase chain reaction, and subcloned into the mammalian expression vector pSVL. The recombinant vector was transfected into the COS cell line by the calcium phosphate technique for transient expression of the scFvs. Immunoaffinity purification (via the C-terminal c-Myc tag) of the scFvs from the cell culture supernatant yielded a protein with molecular weight 28.5 kDa (corresponding to the molecular weight of the scFvs). Two other protein contaminants co-purified with the scFvs, which were tentatively identified by molecular weight as host c-Myc proteins. Purification of the scFvs will allow a comparison of the scFvs produced by mammalian cells and bacteria with respect to the affinity of the scFvs for antigen.

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a Biochemistry Department, Prince of Songk/a University, Hat Yai, Songkla, Thailand.
b Biotechnology Department, University of New South Wales, Sydney, Australia 2052.

Received 30 January, 1997