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.

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble β-galactosidase, the gene of Thermus thermophilus KNOUC112 β-galactosidase (KNOUC112 β-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 β-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The β-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion βgalactosidase expressed via pThioHis C at 37°C was about 5 times higher than that of unfused β-galactosidase expressed via pET-5b at 37°C. Inclusion body of β-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 β-gal was expressed via pET-5b at 37°C. Fusion β-galactosidase expressed at 37°C via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25°C prevented the formation of inclusion body, optimally at 25°C. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25°C. The soluble production of Thermus thermophilus KNOUC112 β-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.