Preparation and characterization of recombinant protein ScFv(CD11c)-TRP2 for tumor therapy from inclusion bodies in Escherichia coli.
Dendritic cells (DCs)-based immunotherapy represents an approach to the prevention and treatment of cancers. Targeting antigens to receptors on DCs can be expected to enhance immune response. We have constructed an expression vector pET32a(+)-ScFv(CD11c)-TRP2 based on a single-chain antibody fragment (ScFv) that targets the high affinity receptor CD11c which is expressed on murine DCs. The 3'-terminal end of the ScFv was ligated to the gene for MHC class I molecule-recognized peptide from mouse tyrosine-related protein 2 (TRP2). Using this vector, we have expressed and purified ScFv(CD11c)-TRP2, a fusion protein that could target TRP2 peptide to CD11c on DCs in vivo to elicit anti-tumor responses. This fusion protein was expressed in inclusion bodies in Escherichia coli BL21(DE3) and was refolded and purified on-column effectively by immobilized metal affinity chromatography using His-tag. Flow cytometry assays showed the specific binding ability of ScFv(CD11c)-TRP2 to DCs, which could be blocked by a hamster anti-mouse CD11c produced by N418 hybridoma. Further studies demonstrated that ScFv(CD11c)-targeted TRP2 peptide processed by DCs was capable of stimulating T cells proliferation. Thus, this fusion protein provides a basis for further research in cancer therapy in vivo.
Kou
Geng
G
International Joint Cancer Institute, Second Military Medical University, New Library Building West 10th-11th Floor, 800 Xiang Yin Road, Shanghai 200433, PR China.
Shi
Shu
S
Wang
Hao
H
Tan
Min
M
Xue
Jingya
J
Zhang
Dapeng
D
Hou
Sheng
S
Qian
Weizhu
W
Wang
Shuhui
S
Dai
Jianxin
J
Li
Bohua
B
Guo
Yajun
Y
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001483:Base Sequence; D039521:CD11c Antigen / Q000235:genetics / Q000627:therapeutic use; D003001:Cloning, Molecular; D017931:DNA Primers; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000627:therapeutic use; D015183:Restriction Mapping
D015703:Antigens, CD; D000970:Antineoplastic Agents; D039521:CD11c Antigen; D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C500290:peptide SVYDFFVWL