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1.
Tetrameric structure of a serine integrase catalytic domain 总被引:1,自引:0,他引:1
The serine integrases have recently emerged as powerful new chromosome engineering tools in various organisms and show promise for therapeutic use in human cells. The serine integrases are structurally and mechanistically unrelated to the bacteriophage lambda integrase but share a similar catalytic domain with the resolvase/invertase enzymes typified by the resolvase proteins from transposons Tn3 and gammadelta. Here we report the crystal structure and solution properties of the catalytic domain from bacteriophage TP901-1 integrase. The protein is a dimer in solution but crystallizes as a tetramer that is closely related in overall architecture to structures of activated gammadelta-resolvase mutants. The ability of the integrase tetramer to explain biochemical experiments performed in the resolvase and invertase systems suggests that the TP901 integrase tetramer represents a unique intermediate on the recombination pathway that is shared within the serine recombinase superfamily. 相似文献
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Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination
Sean D. Colloms Christine A. Merrick Femi J. Olorunniji W. Marshall Stark Margaret C. M. Smith Anne Osbourn Jay D. Keasling Susan J. Rosser 《Nucleic acids research》2014,42(4):e23
Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by ϕC31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. ϕC31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition. 相似文献
3.
The Mycobacterium tuberculosis prophage-like element phiRv1 encodes a site-specific recombination system utilizing an integrase of the serine recombinase family. Recombination occurs between a putative attP site and the host chromosome, but is unusual in that the attB site lies within a redundant repetitive element (REP13E12) of which there are seven copies in the M. tuberculosis genome; four of these elements contain attB sites suitable for phiRv1 integration in vivo. Although the mechanism of directional control of large serine integrases is poorly understood, a recombination directionality factor (RDF) has been identified that is required for phiRv1 integrase-mediated excisive recombination in vivo. Here we describe defined in vitro recombination reactions for both phiRv1 integrase-mediated integration and excision and show that the phiRv1 RDF is not only required for excision but inhibits integrative recombination; neither reaction requires DNA supercoiling, host factors, or high-energy cofactors. Integration, excision and excise-mediated inhibition of integration require simple substrates sites, indicating that the control of directionality does not involve the manipulation of higher-order protein-DNA architectures as described for the tyrosine integrases. 相似文献
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Rao CV 《Current opinion in biotechnology》2012,23(5):689-694
Despite substantial progress in synthetic biology, we still lack the ability to engineer anything as complex as Nature has. One of the many reasons is that we lack the requisite tools for independently controlling the expression of multiple genes in parallel. While our toolbox is still spare, the situation is rapidly changing. This opinion discusses some recent approaches and open challenges in designing orthogonal regulators of gene expression in bacteria. 相似文献
6.
Cagliari CI De Caroli FP Nakahata AM Araújo MS Nakaie CR Sampaio MU Sampaio CA Oliva ML 《Biochemical and biophysical research communications》2003,311(1):241-245
The kallikrein inhibitor found in Bauhinia bauhinioides seeds (BbKI) differs from classical Kunitz plant inhibitors in the lack of disulfide bridges in its structure [Biochim. Biophys. Acta 1477 (2000) 64-74]. In this study, we examined whether structural properties may be involved in inhibitory specificity and, if so, whether those properties might be useful tools in designing compounds that interfere with enzyme activity. Peptides structurally related to the BbKI (RPGLPVRFESPLRINIIKE-NH(2)) reactive site were synthesized by solid-phase method and assayed for serine proteinase activity. The peptides RPGLPVRFESPLRINIIKE-NH(2), RPGLPVRFESPL-NH(2), and GLPVRFES-NH(2) were efficient tissue kallikrein inhibitors, with I(50) values of 0.54 microM, 0.87 microM, and 0.5mM, respectively. The lasting inhibitory effect was observed in incubation periods of up to 120 min. None of the studied peptides interfere with the activity of thrombin, factor Xa or trypsin, although the native protein BbKI is a potent trypsin inhibitor. 相似文献
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Daniel Gromadzki 《Reviews in Environmental Science and Biotechnology》2010,9(4):301-306
Nanostructured amphiphilic block copolymers, graft copolymers, polymeric thermally responsive molecular brushes and polymer
stars are only few examples of macromolecular architectures accessible either via controlled/living radical polymerization
(CLRP) techniques or the combination of CLRP mechanisms with efficient post-polymerization routes including click chemistry.
Precise control over the composition, molecular weight and functionalities is a prerequisite for soft polymeric materials
to self-organize into ordered morphologies. This contribution describes novel orthogonal chemical routes for the synthesis
of macromolecular architectures and self-assembly of functional soft polymeric materials. Emerging potential applications
of well-defined block and graft copolymers are outlined as well. 相似文献
11.
Granzymes (lymphocyte serine proteases): characterization with natural and synthetic substrates and inhibitors 总被引:11,自引:0,他引:11
Natural killer (NK) and cytotoxic T-lymphocytes (CTLs) kill cells within an organism to defend it against viral infections and the growth of tumors. One mechanism of killing involves exocytosis of lymphocyte granules which causes pores to form in the membranes of the attacked cells, fragments nuclear DNA and leads to cell death. The cytotoxic granules contain perforin, a pore-forming protein, and a family of at least 11 serine proteases termed granzymes. Both perforin and granzymes are involved in the lytic activity. Although the biological functions of most granzymes remain to be resolved, granzyme B clearly promotes DNA fragmentation and is directly involved in cell death. Potential natural substrates for Gr B include procaspases and other proteins involved in cell death. Activated caspases are involved in apoptosis. The search continues for natural substrates for the other granzymes. The first granzyme crystal structure remains to be resolved, but in the interim, molecular models of granzymes have provided valuable structural information about their substrate binding sites. The information has been useful to predict the amino acid sequences that immediately flank each side of the scissile peptide bond of peptide and protein substrates. Synthetic substrates, such as peptide thioesters, nitroanilides and aminomethylcoumarins, have also been used to study the substrate specificity of granzymes. The different granzymes have one of four primary substrate specificities: tryptase (cleaving after Arg or Lys), Asp-ase (cleaving after Asp), Met-ase (cleaving after Met or Leu), and chymase (cleaving after Phe, Tyr, or Trp). Natural serpins and synthetic inhibitors (including isocoumarins, peptide chloromethyl ketones, and peptide phosphonates) inhibit granzymes. Studies of substrate and inhibitor kinetics are providing valuable information to identify the most likely natural granzyme substrates and provide tools for the study of key reactions in the cytolytic mechanism. 相似文献
12.
In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1-mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNA(CUA)-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from approximately 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo. 相似文献
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L I Pizer 《Biochimica et biophysica acta》1966,124(2):418-420
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P Cherepanov W Pluymers A Claeys P Proost E De Clercq Z Debyser 《FASEB journal》2000,14(10):1389-1399
A synthetic gene encoding for HIV-1 integrase was designed to circumvent the intrinsic instability and the repressor elements present in the wild-type gene. High-level expression of HIV-1 integrase was obtained in various human cell lines independently of viral accessory proteins. A human 293T cell line was selected that stably expresses HIV-1 integrase and has growth kinetics comparable to the parental cell line. The enzyme was localized in the nucleus and remained stably associated with the chromosomes during mitosis. Lentiviral vector particles carrying the inactivating D64V mutation in the integrase gene were capable of stably transducing 293T cells when complemented in the producer cells with integrase expressed from the synthetic gene. When the cell line that stably expresses integrase was infected with the defective viral particles, complementation of integrase activity was detected as well. Expression of active HIV-1 integrase in human cells will facilitate the study of the interplay between host and viral factors during integration. 相似文献
15.
J. K. Tripp 《Zoo biology》1985,4(3):225-234
The purpose of this research was to explore the impact of the provision of manipulable and edible materials on the activity level of captive orangutans. The effects of three environmental conditions—bare exhibit, manipulables present, and manipulables plus edibles present—on the gross motor activity of four captive orangutans were compared using a multielement design. Results indicated that activity increased under the enriched conditions with increases in activity type specific to the environmental change. In addition, zoo visitors rated the exhibit more favorably on days the enriched conditions were in effect than on bare exhibit days. 相似文献
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Athanassios Stavrakoudis Ioannis N. Demetropoulos Constantinos Sakarellos Maria Sakarellos-Daitsiotis Vassilios Tsikaris 《International journal of peptide research and therapeutics》1997,4(4-6):481-487
Summary The design, synthesis and catalytic properties of a cyclic branched peptide carrier that possesses the catalytic triad residues
of the serine proteases is reported. The synthesis of the peptide model was totally completed on solid support using three
different orthogonal amino protecting groups. Hydrolytic activity measurements against Suc-Ala-Ala-Ala-pNA substrate showed
that it is hydrolysed by the peptide model to a small extent. Despite this small hydrolytic activity, it is the first time,
to our knowledge, that hydrolysis of such a substrate is reported by an enzyme model compound. Contrary, this enzyme model
peptide showed considerable activity against the Boc-Ala-pNP substrate (k
cat
=0.414 min−1 andK
m
=0.228 mm). These results suggest that the designed carrier brings in appropriate contact the catalytic triad residues (Ser,
His, Asp) resulting in the obtained hydrolytic activity. 相似文献
17.
Athanassios Stavrakoudis Ioannis N. Demetropoulos Constantinos Sakarellos Maria Sakarellos-Daitsiotis Vassilios Tsikaris 《Letters in Peptide Science》1997,4(4-6):481-487
The design, synthesis and catalytic properties of acyclic branched peptide carrier that possesses thecatalytic triad residues of the serine proteases isreported. The synthesis of the peptide model wastotally completed on solid support using threedifferent orthogonal amino protecting groups.Hydrolytic activity measurements againstSuc-Ala-Ala-Ala-pNA substrate showed that it ishydrolysed by the peptide model to a small extent.Despite this small hydrolytic activity, it is thefirst time, to our knowledge, that hydrolysis of such a substrate is reported by an enzyme model compound.Contrary, this enzyme model peptide showedconsiderable activity against the Boc-Ala-pNPsubstrate (kcat = 0.414 min–1 and Km = 0.228 mm). These results suggest that thedesigned carrier brings in appropriate contact thecatalytic triad residues (Ser, His, Asp) resulting inthe obtained hydrolytic activity. 相似文献
18.
Advances in synthetic biology are contributing to diverse research areas, from basic biology to biomanufacturing and disease
therapy. We discuss the theoretical foundation, applications, and potential of this emerging field. 相似文献
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Allyson Lister Varodom Charoensawan Subhajyoti De Katherine James Sarath Chandra Janga Julian Huppert 《Genome biology》2009,10(6):309-3
A report of BioSysBio 2009, the IET conference on Synthetic Biology, Systems Biology and Bioinformatics, Cambridge, UK, 23-25 March 2009. 相似文献