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

Abstract

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.

Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas

“…Restriction digestion with SfiI and NotI endonucleases, and agarose gel purification of the digested linked product, preceded ligation of this DNA into the SfiI-and NotIdigested pRSET-Angiogenin SfiI/NotI vector (Figure 1). This vector was constructed from the pRSET SfiI/NotI vector (Yi et al, 1999) by PCR. The PCR protocol used the forward primer 5'-GTTAT-CCT GGCT GCCGCCTCCACCAGAGCCACCTCCGCCCGATCCG-CCACCGCCTGCGGCCGCCCG-3' and the back primer 5'-CGTCCGTAAGCTTGATCCGGCTGCTAACAAAGCC-3' for 30 cycles of 1 min at 958C, 2 min at 558C, and 1 min at 728C.…”

“…The SC142 scFv gene fragment, a fusion protein of predicted size in SDS -PAGE and Western blot analyses and found to be positive in ELISA and confirmed by DNA sequencing, was subcloned into pRSET SfiI/NotI vector (Yi et al, 1999) and transformed into E. coli BL21 (DE3) cells for SC142 scFv purification.…”

“…From (Yi et al, 1999) was amplified with the primers as described in Materials and Methods. The vector was modified to include a HindIII site and angiogenin site after NotI site.…”

Inhibition of the EGF-induced activation of phospholipase C-γ1 by a single chain antibody fragment

“…When F7-scFv was expressed in E. coli BL21 using pRSET S®I/NotI prokaryotic expression vector (Yi et al, 1999) inclusion bodies which was purely composed of F7-scFv were formed. To obtain soluble F7-scFv, the inclusion bodies were denatured using guanidine HCl and renatured by dialysis.…”

“…To obtain soluble F7-scFv, the inclusion bodies were denatured using guanidine HCl and renatured by dialysis. The soluble F7-scFv bound to puri®ed PLC-g1, while CEA79-scFv, which recognizes carcinoembryonic antigen (Yi et al, 1999), did not (Figure 3a). After mixing the soluble F7-scFv with COS-7 cell lysate, PLC-g1 was precipitated with anti-PLC-g1 antibody, which recognizes the 10 amino acids at the C-terminus of PLC-g1 (RTRVNGDNRL).…”