We have engineered the phage displayed TEM-1 beta-lactamase to generate enzymes that can be used in homogeneous immunoassays because their activity can be modulated by binding to monoclonal antibodies (Mabs) raised against an unrelated protein. Random peptide libraries were genetically inserted into three loops to create hybrid enzymes with binding sites for Mabs. Insertion points were chosen to be close enough to the active site that complex formation could affect the activity. The antibiotic resistance provided by the beta-lactamase activity was used to select the clones encoding active enzymes. Biopanning of the active libraries on immobilized Mabs against the prostate specific antigen (PSA) or on streptavidin yielded enzymes with binding sites for these proteins. Their activity could be regulated by Mab or streptavidin binding. The dissociation constants of the complexes are in the 10(-9) to 10(-6) M range. In a competitive assay, PSA could be detected at a minimal concentration of 10(-9) M. The Mabs recognize mimotopes as no sequence similarity was found between inserts in regulated clones and fragments of the PSA sequence. The method can be developed to generate signaling molecules to be used for the detection of analytes in solution without identification of the epitope.
The nonstructural (NS) transcription unit of minute virus of mice (MVMp) encodes proteins that are involved in viral DNA replication and in the regulation of homologous and heterologous promoters. Moreover, it has been shown that NS-protein accumulation is toxic for transformed cells. With the aim of identifying the NS-protein function(s) responsible for cytotoxicity, point mutations and deletions were introduced in the NS-protein-coding sequence of MVMp. This strategy indicated that in transformed human NBE cells, the NS-1 protein is indispensable for MVMp DNA replication, trans activation of the late parvoviral promoter P38, trans inhibition of the long terminal repeat promoter of the Rous sarcoma virus, and cytotoxicity. Moreover, some mutations led to the dissociation of the replicative and regulatory functions of the NS-1 protein and showed that cytotoxicity correlated with the latter, more particularly with the capacity to trans inhibit the heterologous promoter. The NS-1 sequences required for cytotoxicity were found to be restricted to the amino- and carboxy-terminal portions of the protein. Although the cytotoxicities of NS-1 extremities were weak when the extremities were tested separately, the cytotoxicities were comparable to that of the full protein when the extremities were fused. Interestingly, an overall negative charge can be predicted from the NS-1 sequence over about 100 amino acids at both ends. The conservation of this charge distribution among the NS proteins of different parvoviruses suggests that NS-1 may bear some similarities to acidic transcriptional activators.
The NS-1 gene of the parvovirus minute virus of mice (MVM) (prototype strain, MVMp) was fused in phase with the sequence coding for the DNA-binding domain of the bacterial LexA repressor. The resulting chimeric protein, LexNS-1, was tested for its transcriptional activity by using various target promoters in which multiple LexA operator sequences had been introduced. Under these conditions, NS-1 was shown to stimulate gene expression driven by the modified long terminal repeat promoters (from the retroviruses mouse mammary tumor virus and Rous sarcoma virus) and P38 promoter (from MVMp), indicating that the NS-1 protein is a potent transcriptional activator. It is noteworthy that in the absence of LexA operator-mediated targeting, the genuine mouse mammary tumor virus and Rous sarcoma virus promoters were inhibited by NS-1. Together these data strongly suggest that NS-1 contains an activating region able to induce promoters with which this protein interacts but also to repress transcription from nonrecognized promoters by a squelching mechanism similar to that described for other activators. Deletion mutant analysis led to the identification of an NS-1 domain that exhibited an activating potential comparable to that of the whole polypeptide when fused to the DNA-binding region of LexA. This domain is localized in the carboxy-terminal part of NS-1 and corresponds to one of the two regions previously found to be responsible for toxicity. These results argue for the involvement of the regulatory functions of NS-1 in the cytopathic effect of this parvovirus product.
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