Expression of GA733-Fc fusion protein as a vaccine candidate for colorectal cancer in transgenic plants.
The tumor-associated antigen GA733 is a cell-surface glycoprotein highly expressed in colorectal carcinomas. In this study, 3 recombinant genes were constructed as follows: GA733 tagged to the ER retention sequence KDEL (GA733K), GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), and GA733-Fc fused to the ER retention sequence (GA733-FcK). Agrobacterium-mediated transformation was used to generate transgenic plants expressing recombinant genes. The presence of transgenes was confirmed by genomic PCR. Western blot, confocal immunofluorescence, and sandwich ELISA showed the expression of recombinant proteins. The stability, flexibility, and bioactivity of recombinant proteins were analyzed and demonstrated through N-glycosylation analysis, animal trials, and sera ELISA. Our results suggest that the KDEL retained proteins in ER with oligomannose glycan structure and enhanced protein accumulation level. The sera of mice immunized with GA733-FcK purified from plants contained immunoglobulins which were at least as efficient as the mammalian-derived GA733-Fc at recognizing human colorectal cancer cell lines. Thus, a plant system can be used to express the KDEL fusion protein with oligomannose glycosylation, and this protein induces an immune response which is comparable to non-KDEL-tagged, mammalian-derived proteins.
Department of Medicine, Medical Research Institute, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea.
Zhe
Z
Lu
Kyung-Jin
KJ
Lee
Yingxue
Y
Shao
Jeong-Hwan
JH
Lee
Yangkang
Y
So
Young-Kug
YK
Choo
Doo-Byoung
DB
Oh
Kyung-A
KA
Hwang
Seung Han
SH
Oh
Yeon Soo
YS
Han
Kisung
K
Ko
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019496:Cancer Vaccines / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000276:immunology / Q000628:therapy*; D004797:Enzyme-Linked Immunosorbent Assay; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D007141:Immunoglobulin Fc Fragments; D009842:Oligopeptides; D011134:Polysaccharides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C073762:lysyl-aspartyl-glutamyl-leucine