RESUMO
A key component of the recently described bioluminescent system of higher fungi is luciferase, a new class of proteins. The properties of fungal luciferase and their relationship with its structure are interesting both for improving autoluminescent systems already created on its basis and for creating new ones. Therefore, it is extremely important to understand the spatial structure of this protein. We have performed heterologous expression and purification of Neonothopanus nambi luciferase, obtained a protein suitable for subsequent crystallization, and also determined some biochemical properties of the recombinant luciferase.
Assuntos
Agaricales/metabolismo , Luciferases/biossíntese , Luciferases/química , Dicroísmo Circular , Detergentes , Escherichia coli/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Cinética , Luminescência , Espectroscopia de Ressonância Magnética , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Saccharomycetales/metabolismo , TemperaturaRESUMO
We overexpressed duplex-specific nuclease (DSN) from Kamchatka crab in Escherichia coli cells and developed procedures for purification, renaturation, and activation of this protein. We demonstrated identity of the properties of the native and recombinant DSN. We also successfully applied the recombinant DSN for full-length cDNA library normalization.
Assuntos
Braquiúros/enzimologia , Endonucleases/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Sequência de Bases , Braquiúros/genética , Braquiúros/metabolismo , Clonagem Molecular/métodos , DNA Complementar/genética , Endonucleases/química , Endonucleases/genética , Ativação Enzimática , Estabilidade Enzimática , Ácidos Nucleicos Heteroduplexes/metabolismo , Dobramento de Proteína , Renaturação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A gene encoding of glutamyl-specific endopeptidase precursor from Bacillus licheniformis has been cloned in Escherichia coli cells. The recombinant protein was expressed and accumulated as cytoplasmic insoluble inclusion bodies. Washed inclusion bodies were solubilized in 6 M guanidine-HCL in the presence of reducing agent. The following precursor renaturation was performed by fast frequent dilution method. The highest yield of the refolded protein was achieved at pH value of 8.5 and 4 degrees C. The renaturation process was accompanied by a gradual splitting of Glu(-48)/Thr(-47) and Glu(-13)/Lys(-12) peptide bonds. A 26-kDa protein proved to be an end product of in vitro renaturation. The mature glutamyl endopeptidase with a molecular mass of 25 kDa was obtained after a limited proteolysis of the 26-kDa protein was performed by subtilisin or trypsin. The 26-kDa protein was purified by gel filtration on a Superdex 75 column. Comparative characteristics of the thermal stability and catalytic properties of the 26-kDa and 25-kDa proteins showed that complete cleavage of the N-terminal pro-peptide by exogenous proteinase is necessary for a final packing and activation of the B. licheniformis glutamyl endopeptidase.
Assuntos
Bacillus/enzimologia , Bacillus/genética , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Sequência de Aminoácidos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Concentração de Íons de Hidrogênio , Corpos de Inclusão/enzimologia , Corpos de Inclusão/genética , Dados de Sequência Molecular , Renaturação Proteica , Serina Endopeptidases/isolamento & purificaçãoRESUMO
We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.
Assuntos
DNA Complementar/química , DNA de Cadeia Simples/química , Biblioteca Gênica , Animais , Braquiúros , Endonucleases/química , Humanos , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
Glu,Asp-specific endopeptidases represent a new subfamily of chymotrypsin-like proteolytic enzymes. These enzymes prefer Glu or Asp residues in the P1 position of the substrates. p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal glutamic or aspartic acid residues have been obtained. Acyl peptide p-nitroanilides were synthesized via acylation of glutamic or aspartic acid p-nitroanilides using methyl esters of the respective N-acylated peptides, generally with good yields. The reactions were performed in organic solvents using subtilisin 72 sorbed on silica as a catalyst. The kinetic parameters for the hydrolysis of these p-nitroanilides with proteinases from Bacillus intermedius and Bacillus licheniformis were determined.
Assuntos
Bioquímica/métodos , Compostos Cromogênicos/síntese química , Compostos Cromogênicos/metabolismo , Serina Endopeptidases/metabolismo , Anilidas/química , Ácido Aspártico/metabolismo , Bacillus/enzimologia , Ácido Glutâmico/metabolismo , Cinética , Especificidade por Substrato , Subtilisinas/metabolismoRESUMO
The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease.