RESUMO
We describe an efficient strategy to produce high-quality proteins by using a single large IMAC chromatography column and enzymatic His-tag removal via the TAGZyme system in pilot scale. Numerous quality assays demonstrated a high purity of the final product, the human cytokine Interleukin-1beta (IL-1beta). The protein preparation was apparently free of host cell proteins, endotoxins, protease, and aggregates. The N-terminal amino acid sequence of IL-1beta was in full agreement with the natural mature form of IL-1beta. The homogeneity of the product was further shown by X-ray structure determination which confirmed the previously solved structure of the protein. We propose the applied workflow as a strategy for industrial production of protein-based biopharmaceuticals.
Assuntos
Biotecnologia/métodos , Interleucina-1beta/biossíntese , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Cromatografia de Afinidade , Eletroforese em Gel Bidimensional , Endodesoxirribonucleases/isolamento & purificação , Endorribonucleases/isolamento & purificação , Endotoxinas/isolamento & purificação , Exopeptidases/isolamento & purificação , Histidina/metabolismo , Humanos , Interleucina-1beta/química , Interleucina-1beta/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de ProteínaRESUMO
The exoprotease from Oenococcus oeni produced in stress conditions was purified to homogeneity in two steps, a 14-fold increase of specific activity and a 44% recovery of proteinase activity. The molecular mass was estimated to be 33.1 kDa by gel filtration and 17 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggest that the enzyme is a dimer consisting of two identical subunits. Optimal conditions for activity on grape juice were 25 degrees C and a pH of 4.5. Incubation at 70 degrees C, 15 min, destroyed proteolytic activity. The SDS-PAGE profile shows that the enzyme was able to degrade the grape juice proteins at a significantly high rate. The activity at low pH and pepstatin A inhibition indicate that this enzyme is an aspartic protease. The protease activity increases at acidic pH suggesting that it could be involved in the wine elaboration.
Assuntos
Exopeptidases/isolamento & purificação , Exopeptidases/metabolismo , Cocos Gram-Positivos/enzimologia , Bebidas , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Cocos Gram-Positivos/crescimento & desenvolvimento , Leuconostoc/enzimologia , Leuconostoc/crescimento & desenvolvimento , Inibidores de Proteases/farmacologia , Rosales , Vinho/microbiologiaRESUMO
SUMO-1 is a ubiquitin-like protein functioning as an important reversible protein modifier. To date there is no report on a SUMO-1 hydrolase/isopeptidase catalyzing the release of SUMO-1 from its precursor or SUMO-1-ligated proteins in mammalian tissues. Here we found multiple activities that cleave the SUMO-1 moiety from two model substrates, (125)I-SUMO-1-alphaNH-HSTVGSMHISPPEPESEEEEEHYC and/or GST-SUMO-1-(35)S-RanGAP1 conjugate, in bovine brain extracts. Of them, a major SUMO-1 C-terminal hydrolase had been partially purified by successive chromatographic operations. The enzyme had the ability to cleave SUMO-1 not only from its precursor but also from a SUMO-1-ligated RanGAP1 but did not exhibit any significant cleavage of the ubiquitin- and NEDD8-precursor. The activity of SUMO-1 hydrolase was almost completely inhibited by N-ethylmaleimide, but not by phenylmethanesulfonyl fluoride, EDTA, and ubiquitin-aldehyde known as a potent inhibitor of deubiquitinylating enzymes. Intriguingly, the apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1. These results indicate that there are multiple SUMO-1 hydrolase/isopeptidases in mammalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central role in processing of the SUMO-1-precursor.
