Despite of membrane catechol-O-methyltransferase (MBCOMT, EC 2.1.1.6) physiological importance on catecholamines' O-methylation, no studies allowed their total isolation. Therefore, for the first time, we compare the performance of three hydrophobic adsorbents (butyl-, epoxy-, and octyl-Sepharose) in purification of recombinant human COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and hMBCOMT's binding and elution conditions. Results show that hMBCOMT's adsorption was promoted on octyl and butyl at ≤375 mM NaH2 PO4, while on epoxy higher concentrations (>850 mM) were required. Additionally, hMBCOMT's elution was promoted on epoxy, butyl, and octyl using respectively 0.1-0.5, 0.25-1, and 1% of Triton X-100. On butyl media, a stepwise strategy using 375 and 0 mM NaH2PO4, followed by three elution steps at 0.25, 0.7 and 1% Triton X-100, allowed selective hMBCOMT isolation. In conclusion, significant amounts of MBCOMT were purified with high selectivity on a single chromatography procedure, despite its elution occurs on multiple peaks. Although successful applications of hydrophobic interaction chromatography in purification of membrane proteins are uncommon, we proved that traditional hydrophobic matrices can open a promising unexplored field to fulfill specific requirements for kinetic and pharmacological trials.
Research on RNA has led to many important biological discoveries and the improvement of therapeutic technologies. In particular, there is a great focus on small RNA and ribosomal RNA owing to their key functions in the cell, which make them excellent therapeutic targets. Although the study of these RNA classes is progressing, some limitations have been found regarding the use of suitable techniques that are able to produce and isolate biologically competent and chemically stable RNA. To address this, we have developed a novel histidine affinity chromatography-based isolation methodology for small and ribosomal RNA molecules. The new procedure involves three main steps: (1) cell lysis with guanidinium buffer, (2) RNA primary isolation with ammonium sulfate precipitation and (3) histidine affinity chromatography to specifically purify small RNA and ribosomal RNA from other Escherichia coli impurities (genomic DNA and proteins). The RNA quality assessment revealed that both RNA species were obtained with a high recovery, integrity and purity. The potential of this method to achieve a reproducible RNA isolation with appropriate quality has been demonstrated and it should have broad application in the structural, biophysical and biomedical investigation of systems involving RNA components.
6S RNA is an abundant non-coding RNA in Escherichia coli (E. coli), but its function has not been discovered until recently. The first advance on 6S RNA function was the demonstration of its ability to bind the s 70 -holoenzyme form of RNA polymerase, inhibiting its activity and consequently the transcription process. The growing interest in the investigation of non-coding small RNAs (sRNA) calls for the development of new methods for isolation and purification of RNA. This work presents an optimized RNA extraction procedure and describes a new affinity chromatography method using a histidine support to specifically purify 6S RNA from other E. coli sRNA species. The RNA extraction procedure was optimized, and a high yield was obtained in the separation of sRNA and ribosomal RNA (rRNA) from total RNA (RNAt). This improved method takes advantage of its simplicity and significant cost reduction, since some complex operations have been eliminated. A purification strategy was also developed to separate 6S RNA from an sRNA mixture. Pure RNA can be advantageously obtained using the histidine-affinity chromatography method, aiming at its application to structural or functional studies.
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