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1.
Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies. 总被引:5,自引:0,他引:5
Bispecific single-chain diabodies (scDb) consist of the variable heavy and light chain domains of two antibodies connected by three linkers. The structure of an scDb in the V(H)-V(L) orientation is V(H)A-linkerA-V(L)B-linkerM-V(H)B-linkerB-V(L)A, with linkers A and B routinely chosen to be 5-6 residues and linker M 15-20 residues. Here, we applied display of scDb on filamentous phage to analyse the composition of optimal linker sequences. The three linkers were randomized in length and sequence using degenerated triplets coding for only six hydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly, Ala). Antigen-binding clones were then isolated by one to two rounds of selection on the two different antigens recognized by the bispecific scDb. Using an scDb directed against carcinoembryonic antigen (CEA) and beta-galactosidase (Gal), we found that monomeric scDb had a preferred length of 15 or more amino acid residues for the middle linker M and of 3-6 residues for the linkers A and B. No obvious bias towards a preferred linker sequence was observed. Reduction of the middle linker below 13 residues led to the formation of dimeric scDb, which most likely results from interchain pairing between all the V(H) and V(L) domains. Dimeric scDb were also formed by fragments possessing a long linker M and linkers A and B of 0 or 1 residue. We assume that these dimeric scDb are formed by intrachain pairing of the central variable domains and interchain pairing of the flanking variable domains. Thus, the latter molecules represent a novel format of bispecific and tetravalent molecules. The described strategy allows for the isolation of both optimized and minimal linker sequences for the assembly of monomeric or dimeric single-chain diabodies. 相似文献
2.
Monoclonal antibodies were produced to electrophoretically pure hydrogenase fromThiocapsa roseopersicina. Protein immunoelectroblotting was used to identify the hydrogenase-specific antibodies. Among the 18 monoclonal antibodies selected by enzyme immunoassay, three were found to react with highly immunogenic trace contaminating proteins. One cell line produced antibody that inhibitied hydrogenase activity. This was the first specific inhibitor of the hydrogenase function. The results suggest that monoclonal antibodies could provide valuable new informations about the enzyme structure as well. 相似文献
3.
Summary Feeding the secondary plant compound 6-Methoxybenzoxazolinone (6-MBOA) during winter to a free living population of Microtus townsendii accelerated breeding in females. Both the recruitment of young and sexual maturation of these young were advanced by four weeks in comparison with a control population. Overwintered females supplied with 6-MBOA matured at a lower body weight than control females. But there was no such weightat-maturity difference between the grids for males. During April and May 72% of all juveniles captured came from the experimental population. Winter reproduction and early recruitment of young stimulated by 6-MBOA could have important population consequences for these voles. 相似文献
4.
5.
Enzymatic activity and filament assembly of Acanthamoeba myosin II are regulated by adjacent domains at the end of the tail 总被引:1,自引:0,他引:1
Polyclonal antibodies raised against a synthetic peptide consisting of the last 19 amino acids at the end of the coiled-coil region of the heavy chains inhibited the actin-activated Mg2+-ATPase activity of myosin II and its ability to form filaments. Antibodies against a synthetic peptide corresponding to the 21 adjacent amino acids at the beginning of the non-helical tailpiece, which include the three regulatory phosphorylatable serines, had no effect on either activity. 相似文献
6.
The growth and differentiation of myeloid hematopoietic cells are regulated by different macrophage and granulocyte inducing proteins, those that induce growth and others that induce differentiation. The proteins that induce differentiation but not those that induce growth bind to double-stranded DNA. We now report that purified myeloid cell differentiation-inducing protein causes single strand breaks (nicks) in double-stranded DNA. This DNA nicking may initiate the changes in gene expression that are required for differentiation. 相似文献
7.
Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells 总被引:16,自引:0,他引:16
S W Wong L R Paborsky P A Fisher T S Wang D Korn 《The Journal of biological chemistry》1986,261(17):7958-7968
We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine. 相似文献
8.
C Giaume C Sahuquillo D Louvard H Korn 《Biology of the cell / under the auspices of the European Cell Biology Organization》1986,57(1):33-38
The possibility of current flow between epithelial cells (MDCK) has been evaluated using intracellular electrophysiological techniques. We report here that a significant electrotonic coupling was found in this material at all ages of culture. This observation contrasts with previous reports that confluent MDCK are not ionically coupled and lack gap junctions. Alternative mechanisms for such coupling are considered, with emphasis on the role of tight junctions, should the absence of gap junctions be confirmed. 相似文献
9.
A. M. Duijvestijn R. Schutte Y. G. Köhler C. Korn E. C. M. Hoefsmit 《Cell and tissue research》1983,231(2):313-323
Rat thymic phagocytic cells were characterized in vitro using various light- and electron-microscopical techniques. Thymic cell suspensions were mechanically prepared and enriched for non-lymphoid cells, which were predominantly phagocytic and of three types. Type I showed acid phosphatase (APh) activity in small granules dispersed throughout the cytoplasm and were mostly Ia antigen-positive, although the Ia membrane label varied in intensity and distribution among individual cells. Only a few cells had endogenous peroxidase activity. The type-I cells could not be clearly distinguished morphologically from type-II or -III cells, and most likely comprise precursors of both these cell types. Type-II were large pale cells with many slender cell processes. These cells had APh activity centrally positioned, were strongly positive for Ia on the cell membrane and were negative for endogenous peroxidase. The cytoplasm frequently contained Birbeck granules, which unequivocally classifies these cells as the in vitro equivalent of the interdigitating cells present in the medullary area of the thymus in situ. Type-III cells were rounded with a smooth or ruffled cell membrane and contained vacuoles and many phagolysosomes. They were strongly positive for APh which was present throughout the cytoplasm. About 50% of these cells were positive for endogenous peroxidase in a pattern resembling resident macrophages. The cells were negative for Ia antigens. Type-III cells mostly likely represent the macrophages found in the cortical area of the thymus. 相似文献
10.
Xenopus 5S RNA genes exhibit a pattern of differential expression during development in which some members (oocyte-type) are transcribed only in oocytes, while others (somatic-type) are expressed in both oocytes and somatic cells. Using cloned DNA probes specific for each gene type, we determined the positions of these genes on Xenopus metaphase chromosomes by in situ hybridization. Somatic-type 5S genes in both X. laevis and X. borealis are located at the distal end of the long arm of only one chromosome (number 9). The oocyte-type 5S RNA genes are found at the distal ends of the long arms of most Xenopus chromosomes, including chromosome 9. Thus, large scale differences in chromosomal location cannot explain the selective expression of these genes, as suggested previously. 相似文献