Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system

Biotechnol Bioeng. 2005 Mar 5;89(5):503-11. doi: 10.1002/bit.20283.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / metabolism
  • Autoradiography
  • Cancer Vaccines / immunology
  • Cell Proliferation
  • Cell-Free System
  • Disulfides / chemistry
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial
  • Granulocyte-Macrophage Colony-Stimulating Factor / chemistry
  • Granulocyte-Macrophage Colony-Stimulating Factor / genetics
  • Granulocyte-Macrophage Colony-Stimulating Factor / metabolism
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Idiotypes
  • Immunoglobulin Light Chains
  • Immunoglobulin Variable Region
  • Lymphoma, B-Cell / therapy*
  • Mice
  • Protein Disulfide-Isomerases / metabolism
  • Protein Folding
  • Protein Structure, Tertiary
  • Proteins / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Vaccines / chemistry*
  • Vaccines / genetics
  • Vaccines / immunology


  • Antibodies, Monoclonal
  • Cancer Vaccines
  • Disulfides
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Idiotypes
  • Immunoglobulin Light Chains
  • Immunoglobulin Variable Region
  • Proteins
  • Recombinant Fusion Proteins
  • Vaccines
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Protein Disulfide-Isomerases