title: Generation and functional characterization of the anti-transferrin receptor single-chain antibody-GAL4 (TfRscFv-GAL4) fusion protein.
abstract: BACKGROUND
  The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Transferrin receptor (TfR) is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and TfR binds and intakes of transferrin-iron complex. We have previously generated an anti-TfR single-chain variable fragments of immunoglobulin (scFv) which were cloned from hybridoma cell line producing antibody against TfR linked with a 20 aa-long linker sequence (G4S)4. In the present study, the anti-TfR single-chain antibody (TfRscFv) was fused to DNA-binding domain of the yeast transcription factor GAL4. The recombinant fusion protein, designated as TfRscFv-GAL4, is expected to mediate the entry of DNA-protein complex into targeted tumor cells.
  
  
  RESULTS
  Fusion protein TfRscFv-GAL4 was expressed in an E. coli bacterial expression system and was recovered from inclusion bodies with subsequent purification by metal-chelate chromatography. The resulting proteins were predominantly monomeric and, upon refolding, became a soluble biologically active bifunctional protein. In biological assays, the antigen-binding activity of the re-natured protein, TfRscFv-GAL4, was confirmed by specific binding to different cancer cells and tumor tissues. The cell binding rates, as indicated by flow cytometry (FCM) analysis, ranged from 54.11% to 8.23% in seven different human carcinoma cell lines. It showed similar affinity and binding potency as those of parent full-length mouse anti-TfR antibody. The positive binding rates to tumor tissues by tissue microarrays (TMA) assays were 75.32% and 63.25%, but it showed weakly binding with hepatic tissue in 5 cases, and normal tissues such as heart, spleen, adrenal cortex blood vessel and stomach. In addition, the re-natured fusion protein TfRscFv-GAL4 was used in an ELISA with rabbit anti-GAL4 antibody. The GAL4-DNA functional assay through the GAL4 complementary conjugation with the GAL4rec-GFP-pGes plasmid to verify the GLA4 activity and GAL4rec-recognized specificity functions. It also shows the complex, TfRscFv-GAL4-GAL4rec-GFP-pGes, could be taken into endochylema to express the green fluorescent protein (GFP) with 8 to 10-fold transfection efficiency.
  
  
  CONCLUSIONS
  Results of our study demonstrated that the biofunctianality of genetically engineered fusion protein, TfRscFv-GAL4, was retained, as the fusion protein could both carry the plasmid of GAL4rec-pGes and bind TfR on tumour cells. This product was able to transfect target cells effectively in an immuno-specific manner, resulting in transient gene expression. This protein that can be applied as an effective therapeutic and diagnostic delivery to the tumor using endogenous membrane transport system with potential widespread utility.
authors.affiliation: Department of Pathology, Nanjing Drum Tower Hospital affiliated Nanjing University Medical School, Nanjing, People's Republic of China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Ye
authors.forename: Heyu
authors.initials: H
authors.lastname: Hu
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Wang
authors.forename: Tong
authors.initials: T
authors.lastname: Lu
authors.forename: Zhiquan
authors.initials: Z
authors.lastname: Hu
authors.forename: Xing
authors.initials: X
authors.lastname: Zeng
authors.forename: Shu
authors.initials: S
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Ping
authors.initials: P
authors.lastname: Lei
authors.forename: Cong-Yi
authors.initials: CY
authors.lastname: Wang
authors.forename: Zhangqun
authors.initials: Z
authors.lastname: Ye
authors.forename: Guanxin
authors.initials: G
authors.lastname: Shen
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018922:HL-60 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D020673:Protein Renaturation; D011990:Receptors, Transferrin / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000473:pathology; D046888:Tissue Array Analysis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D057127:Single-Chain Antibodies; D014157:Transcription Factors; D049452:Green Fluorescent Proteins