RESUMO
Halocins, the proteinaceous antimicrobial agents produced by haloarchaea, may be used for the preservation of salted foods and the treatment of diseases. For their application and function explanation, it is necessary to produce the active recombinants. In this work, a haloarchaeal strain producing halocin was isolated from the salt-fermented shrimp and identified as Natrinema sp. RNS21 by 16S rRNA gene sequence analysis. From 1 L of RNS21 culture, about 0.32 mg of halocin with 96% purity was obtained. Based on the molecular weight, stability and amino acid sequence alignment, the antimicrobial peptide belonged to the halocin C8 (HalC8) family. HalC8 was expressed by fusion with glutathione-S-transferase (GST) in E. coli, followed by affinity purification and enterokinase (EK) cleavage. About 6.2 mg of recombinant HalC8 with 95% purity was obtained from 1 L of E. coli culture. MALDI-TOF-MS and RP-HPLC analysis indicated that the molecular weight and folding pattern of purified recombinant HalC8 were the same as those of native HalC8. Recombinant HalC8 showed obvious inhibitory activity against Haloferax volcanii. Contrast to native HalC8, the active recombinant HalC8 could be easily produced in a short time with a high yield.
Assuntos
Escherichia coli , Halobacteriaceae , Escherichia coli/genética , Peptídeos Antimicrobianos , RNA Ribossômico 16S , Halobacteriaceae/genéticaRESUMO
A specific type II restriction endonuclease T.Smu451I has been purified to electrophoretic homogeneity from the frozen cells of soil bacterium Sphingobacterium multivorum 451 (formerly Flavobacterium multivorum 451), using ultrasonic grinding, nucleic acid removal by streptomycin sulfate, protein precipitation by ammonium sulfate and phosphocellulose P-11, DEAE-Cellulose DE-52, Hepharin-Sepharose CL-6B chromatography, and elucidated several characteristics of T.Smu451I. The molecular weight of the enzyme determined by gel filtration and SDS-polyacrylamide gel electrophoresis was calculated to be 45,000 ± 2000 D (dimer) and 23,000 ± 1000 D (monomer), respectively. The isoelectric point (pI) of T.Smu451I is 5.4. T.Smu451I recognizes pentanucleotide palindromic sequences 5'-GGNC↓C-3' and cleaves between C and C in position shown by arrow to produce 3'-cohesive terminus of trinucleotide. Therefore, T.Smu451I is a neoschizomer of T.AsuI.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II , Sphingobacterium , Cromatografia em Gel , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Sphingobacterium/enzimologia , Especificidade por SubstratoRESUMO
Bacillus circulans 528 produces a restriction endonuclease, Bci528I which is an isoschizomer of EcoRI. We purified the enzyme, using Sephadex G-150, Phospho-cellulose, DEAE-cellulose, Hepharin-Sepharose CL-6B chromatography. The specific activity of Bci528I was 29,400 U/mg·protein. Bci528I recognizes 5'-GAATTC-3' in dsDNA and cleaves between G and A of the recognition sequence, producing a symmetric four base 5'overhang.