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1.
J Chromatogr A ; 1109(1): 80-5, 2006 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-16517243

RESUMO

Monoliths are attractive stationary phases for purification of large biomolecules like proteins because of their flow-unaffected properties. Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step. In this work, we investigated properties of commercial metal-chelate methacrylate monoliths-Convective Interaction Media (CIM). Analytical CIM disk monolithic columns and CIM 8 ml monolithic columns were used for purification of tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with 6 histidine tag (GFP-6His). In both cases, purity over 90% was achieved. Dynamic binding capacity at 10% of breakthrough was around 17-18 mg/ml for LK-801 and around 30 mg/ml for GFP-6His. Adsorption isotherm revealed that the maximal capacity is achieved at protein concentration above 60 microg/ml. Dynamic binding capacity and resolution were found to be flow unaffected.


Assuntos
Quelantes/química , Metais/química , Metacrilatos/química , Adsorção , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cobre/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/isolamento & purificação , Fator de Necrose Tumoral alfa/isolamento & purificação
2.
Folia Microbiol (Praha) ; 49(2): 151-5, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15227787

RESUMO

An in vitro study in dual-flow continuous-culture fermentors was conducted with two different concentrations of monensin, cinnamaldehyde or garlic extract added to 1:1 forage-to-concentrate diet in order to determine their effects on selected rumen bacterial populations. Samples were subjected to total DNA extraction, restriction analysis of PCR amplified parts of 16S rRNA genes (ARDRA) and subsequent analysis of the restriction profiles by lab-on-chip technology with the Agilent's Bioanalyser 2100. Eub338-BacPre primer pair was used to select for the bacteria from the genera Bacteroides, Porphyromonas and Prevotella, especially the latter representing the dominant Gram-negative bacterial population in the rumen. Preliminary results of HaeIII restriction analysis show that the effects of monensin, cinnamaldehyde and garlic extract on the BacPre targeted ruminal bacteria are somewhat different in regard to targeted populations and to the nature of the effect. Garlic extract was found to trigger the most intensive changes in the structure of the BacPre targeted population. Comparison of the in silico restriction analysis of BacPre sequences deposited in different DNA databanks and of the results of performed amplified ribosomal DNA restriction analysis showed differences between the predicted and obtained HaeIII restriction profiles, and suggested the presence of novel, still unknown Prevotella populations in studied samples.


Assuntos
Acroleína/análogos & derivados , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Extratos Vegetais/farmacologia , Rúmen/microbiologia , Acroleína/farmacologia , Animais , Antibacterianos/farmacologia , Bactérias/isolamento & purificação , Bacteroides/efeitos dos fármacos , Bacteroides/crescimento & desenvolvimento , Bacteroides/isolamento & purificação , Impressões Digitais de DNA , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , DNA Ribossômico/análise , DNA Ribossômico/isolamento & purificação , DNA Ribossômico/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Alho , Genes Bacterianos , Genes de RNAr , Monensin/farmacologia , Porphyromonas/efeitos dos fármacos , Porphyromonas/crescimento & desenvolvimento , Porphyromonas/isolamento & purificação , Prevotella/efeitos dos fármacos , Prevotella/crescimento & desenvolvimento , Prevotella/isolamento & purificação , RNA Ribossômico 16S/genética , Ribotipagem
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