RESUMO
Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (Kd = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ligação a DNA/química , Proteínas de Membrana/química , Proteínas Oncogênicas Virais/química , Domínios PDZ/genética , Biblioteca de Peptídeos , Proteínas Repressoras/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 1 Homóloga a Discs-Large , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HeLa , Papillomavirus Humano 16/química , Papillomavirus Humano 18/química , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins selected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA-binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.
Assuntos
Proteínas de Ligação a DNA/genética , Genes bcl-2/genética , Subunidades Proteicas/química , Proteínas Repressoras/metabolismo , Sítio de Iniciação de Transcrição , Proteínas Virais/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/genética , Análise de Sequência de Proteína , Proteínas Virais/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
A single-chain derivative of the lambda Cro repressor (scCro) has been randomly mutated in amino acid residues critical for specific DNA recognition to create libraries of protein variants. Utilizing phage display-afforded affinity selection, scCro variants have been isolated for binding to synthetic DNA ligands. Isolated scCro variants were analyzed functionally, both in fusion with phage particles and after expression of the corresponding free proteins. The binding properties with regard to specificity and affinity in binding to different DNA ligands were investigated by inhibition studies and determination of equilibrium dissociation constants for formed complexes. Variant proteins with altered DNA-sequence specificity were identified, which favored binding of targeted synthetic DNA sequences over a consensus operator sequence, bound with high affinity by wild-type Cro. The specificities were relatively modest (2-3-fold, as calculated from K(D) values), which can be attributed to the inherent properties in the design of the selection system; one half-site of the synthetic DNA sequences maintains the consensus operator sequence, and one "subunit" of the variant single-chain Cro dimers was conserved as wild-type sequence. The anticipated interaction between the wild-type subunit and the consensus DNA half-site of target DNA ligands is, hence, expected to contribute to the overlap in sequence discrimination. The binding affinity for the synthetic DNA sequences, however, was improved 10-30-fold in selected variant proteins as compared to "wild-type" scCro.
