Expression and characterization of anti-NCA-95 scFv (CEA 79 scFv) in a prokaryotic expression vector modified to contain a Sfi I and Not I site.
The CEA 79 antibody has been used in bone marrow scintigraphy for the differential diagnosis of skeletal tumors and the evaluation of the bone marrow status of patients with various hematological disorders. The specific localization of radio-labeled CEA 79 antibody in bone marrow depends on its reactivity with NCA-95 (nonspecific cross-reacting antigen-95) present on the surface and in the cytosol of human granulocytes and myelopoietic cells. To make a CEA 79 scFv molecule that would be less immunogenic and more penetrating than the intact mouse immunoglobulin, we constructed a pRSET Sfi I/Not I expression vector. The scFv gene was then excised from a pCANTAB 5 E phage display vector by digestion with Sfi I and Not I and inserted into the pRSET Sfi I/Not I expression vector. Upon transformation of a BL21(DE3)pLysS strain of E. coli, CEA 79 scFv became expressed in inclusion bodies requiring a renaturation process for solubilization. The final yield of CEA 79 scFv was 5 mg per a liter of culture. The refolded CEA 79 scFv exhibited an affinity (Kd = 2.1 x 10(-9) M) equivalent to that of the original CEA 79 antibody (K(d) = 3.3 x 10(-9) M) and the same immunoreactivity to CEA and NCA-95 in Western blots and in immunohistochemical staining experiments.
Yi
K
K
Department of Life Science, Pohang University of Science and Technology, Korea.
Chung
J
J
Kim
H
H
Kim
I
I
Jung
H
H
Kim
J
J
Choi
I
I
Suh
P
P
Chung
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000918:Antibody Specificity; D001483:Base Sequence; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D017931:DNA Primers / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006098:Granulocytes / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016719:Radioimmunodetection
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D017931:DNA Primers; D007135:Immunoglobulin Variable Region; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases