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Rapid expression of vaccine proteins for B‐cell lymphoma in a cell‐free system
Author(s) -
Yang Junhao,
Kanter Gregory,
Voloshin Alexei,
MichelReydellet Nathalie,
Velkeen Hendrik,
Levy Ronald,
Swartz James R.
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20283
Subject(s) - fusion protein , cell free protein synthesis , microbiology and biotechnology , chemistry , iodoacetamide , idiotype , protein disulfide isomerase , antibody , biochemistry , immunoglobulin light chain , cell fusion , cysteine , cell , biology , monoclonal antibody , recombinant dna , protein biosynthesis , disulfide bond , immunology , gene , enzyme
The idiotype (Id)‐granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) fusion proteins are potential vaccines for immunotherapy of B‐cell lymphoma. In this study, four vaccine candidates were constructed by fusing murine GM‐CSF to the amino‐ or carboxy‐terminus of the 38C13 murine B‐lymphocyte Id scFv with two different arrangements of the variable regions of the heavy chain and light chain (VL‐VH and VH‐VL). scFv (VH‐VL) and GM‐CSF/scFv fusion proteins were expressed in an Escherichia coli cell‐free protein synthesis system. In order to promote disulfide bond formation during cell‐free expression, cell extract was pretreated with iodoacetamide (IAM), and a sulfhydryl redox buffer composed of oxidized and reduced glutathione was added. The E. coli periplasmic disulfide isomerase, DsbC, was also added to rearrange incorrectly formed disulfide linkages. The 38C13 B‐lymphocyte Id scFv was expressed with 30% of its soluble yield in active form (43 μg/ml) when tested with an anti‐idiotypic mAb, S1C5, as the capture antibody in radioimmunoassay. It was found that the amino‐terminal GM‐CSF fusion proteins, GM‐VL‐VH and GM‐VH‐VL, showed much higher activity than the carboxy‐terminal GM‐CSF fusion proteins, VL‐VH‐GM and VH‐VL‐GM, in stimulating the cell proliferation of a GM‐CSF‐dependent cell line, NFS‐60. Between the two amino‐terminal GM‐CSF fusion proteins, GM‐VL‐VH showed a higher total and soluble yield than GM‐VH‐VL. © 2005 Wiley Periodicals, Inc.