Expression of a bifunctional chimeric protein A-Vargula hilgendorfii luciferase in mammalian cells.

We have designed and constructed a novel chimeric protein that consisted of a single domain of protein A and luciferase derived from sea-firefly Vargula hilgendorfii with the goal of obtaining a heterofunctional immunological tool. The structural gene of luciferase was fused to the 3' terminus of the D domain gene of protein A with/without a short linker of five amino acids. The resulting constructs under the transcriptional regulation of the Rous sarcoma virus (RSV) promoter, were expressed transiently in simian COS-1 and stably in Chinese hamster ovary (CHO) cells. The properties of the resultant chimeric protein were characterized. The results indicated that the dual properties of the chimeric protein could be retained only after the introduction of a linker of (Gly)4 Ser between the two conjugated moieties. Moreover, the chimeric protein was found to retain at least 50% of the specific activity as compared with the non-fused luciferase. The future prospect of the usage of this chimeric protein in the field of diagnostics was further evaluated by performing bioluminescent immunoassays.

[1]  S. Bottomley,et al.  The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy. , 1994, Protein engineering.

[2]  R. Douthart,et al.  Gene synthesis and functional expression of a protein exhibiting monodomain IgG Fc binding. , 1994, Protein engineering.

[3]  W. Nasser,et al.  Pectate lyase from Bacillus subtilis: molecular characterization of the gene, and properties of the cloned enzyme , 1993, FEBS letters.

[4]  T Prospero,et al.  "Diabodies": small bivalent and bispecific antibody fragments. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[5]  H. Will,et al.  Generation of recombinant CHO(dhfr-) cell lines by single selection for dhfr+ transformants. , 1992, Analytical biochemistry.

[6]  S. Yagi,et al.  Antigen Responsive Antibody–Receptor Kinase Chimera , 1992, Bio/Technology.

[7]  S. Nagata,et al.  Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells. , 1990, Gene.

[8]  H. Okayama,et al.  High-efficiency transformation of mammalian cells by plasmid DNA. , 1987, Molecular and cellular biology.

[9]  B Guss,et al.  Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications. , 1984, The Journal of biological chemistry.

[10]  M. Neuberger.  Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells. , 1983, The EMBO journal.

[11]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[12]  C. Stevens,et al.  Some properties of luciferase from the bioluminescent crustacean, Cypridina hilgendorfii. , 1974, Biochemistry.

[13]  U. K. Laemmli,et al.  Cleavage of structural proteins during , 1970 .

[14]  O. Shimomura,et al.  Cypridina Bioluminescence: Light-Emitting Oxyluciferin-Luciferase Complex , 1969, Science.

[15]  J. W. Hastings,et al.  Total Quantum Flux of Isotropic Sources , 1963 .

[16]  A. M. Chase.  Effects of hydrogen ion concentration and of buffer systems on the luminescence of the Cypridina luciferin-luciferase reaction. , 1948, Journal of cellular and comparative physiology.