Preparation and characterization of two human carcinoembryonic antigen family proteins of neutrophils, CD66b and c, in silkworm larvae.
As a step to investigate the cell adhesion mechanism and physiological roles of two CD66 antigens in human neutrophils, carcinoembryonic antigen gene family member 6 (CGM6, CD66b) and nonspecific cross-reacting antigen (NCA, CD66c), we prepared their soluble recombinant forms in silkworm larvae. Each cDNA fragment for CGM6 and NCA was ligated into the transfer vector pBK283 after modification to encode the protein lacking the membrane anchor. The resultant vectors were introduced to the Bombyx mori nuclear polyhedrosis virus, with which silkworm larvae were infected. Recombinant proteins secreted into the hemolymph of larvae at concentrations up to 1.3 mg/ml were purified by cation exchange followed by gel filtration or antibody affinity chromatography. The smaller apparent masses of the antigens compared with those of the native antigens appeared to be primarily due to incomplete glycosylation. Both recombinant antigens are quite similar to the corresponding native antigens in terms of the antigenic reactivity against a panel of CD66 monoclonal antibodies. In addition, the recombinant CGM6 and NCA exhibited cell binding activity against CHO cells expressing NCA and CGM6, respectively. Thus the two biologically active recombinant CD66 antigens prepared in large quantities in silkworm larvae should be useful for their functional studies, and our present system will be available for the production and purification of other carcinoembryonic antigen family members, whose biological functions are also unknown.
Yamanaka
T
T
Department of Biochemistry, School of Medicine, Fukuoka University, Japan.
Kuroki
M
M
Kinugasa
T
T
Matsuo
Y
Y
Matsuoka
Y
Y
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D012831:Bombyx / Q000196:embryology / Q000235:genetics / Q000276:immunology; D016466:CHO Cells / Q000737:chemistry; D002272:Carcinoembryonic Antigen / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004274:DNA, Recombinant / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006458:Hemolymph / Q000737:chemistry; D006801:Humans; D007814:Larva / Q000276:immunology; D051379:Mice; D009504:Neutrophils / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D015703:Antigens, CD; D000943:Antigens, Differentiation; C069847:CD66 antigens; D002272:Carcinoembryonic Antigen; C486183:Ceacam1 protein, mouse; D015815:Cell Adhesion Molecules; D004274:DNA, Recombinant; D011994:Recombinant Proteins