Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system.
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 microg/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.
Yang
Junhao
J
Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
Kanter
Gregory
G
Voloshin
Alexei
A
Michel-Reydellet
Nathalie
N
Velkeen
Hendrik
H
Levy
Ronald
R
Swartz
James R
JR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001345:Autoradiography; D019496:Cancer Vaccines / Q000276:immunology; D049109:Cell Proliferation; D002474:Cell-Free System; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D016393:Lymphoma, B-Cell / Q000628:therapy*; D051379:Mice; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014612:Vaccines / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D019496:Cancer Vaccines; D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014612:Vaccines; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019704:Protein Disulfide-Isomerases