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 mug/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. (C) 2005 Wiley Periodicals, Inc.