Molecular cloning, expression, purification and characterization of thioredoxin from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178

Abstract

Thioredoxin (Trx) is a highly conserved and multi-functional protein that plays a pivotal role in maintaining the redox state of the cell and in protecting the cell against oxidative stress. Trx gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178 was cloned and expressed as soluble protein in Escherichia coli (designated as PsTrx). Trx gene consisted of an open reading frame of 324-bp nucleotides encoding a protein of 108 amino acids with a calculated molecular mass of 11.88 kDa. The deduced protein included the conserved Cys-Gly-Pro-Cys active-site sequence. After purification by a single step Ni-NTA affinity chromatography, recombinant PsTrx with a high specific activity of 96.67 U/mg was obtained. The purified PsTrx had an optimal temperature and pH of 25 °C and 7.0, respectively, and showed about 55 % of the residual catalytic activity even at 0-10 °C. It had high tolerance to a wide range of NaCl concentrations (0-2 M NaCl) and was stable in the presence of H2O2. This research suggested that PsTrx displayed unique catalytic properties.

Comparative Proteomic Analysis of Psychrophilic vs. Mesophilic Bacterial Species Reveals Different Strategies to Achieve Temperature Adaptation

“…In this regard, two isoforms of the enzyme SOD (Spots 993 and 996), the protein thiorredoxin (Spot 1301) and DNA-Dps (Spot 1126), were induced in P. frigidicola at warm temperatures. The first protein plays a pivotal role in maintaining the redox state of the cell and protects it from oxidative stress ( Wang et al, 2013 ; Lu and Holmgren, 2014 ). DNA-Dps, identified also in S. frigidimarina , is highly conserved and operates, as indicated before, against several kinds of stressors ( Jeong et al, 2006 ; Karas et al, 2015 ) in E. coli , including oxidative damage to protect DNA.…”

Expressão e caracterização enzimática da Tioredoxina redutase (Trr1) e do seu substrato Tioredoxina (Trx1) e identificação de novas drogas por modelagem molecular do alvo Trr1 do fungo patogênico Cryptococcus neoformans

“…2013 PsTrx com a adição de His, foi purificada utilizando cromatografia de afinidade Ni-NTA em resina. As proteínas foram eluídas numa forma gradual, com 5 volumes de resina de 40 e 100 mM de tampão de eluição com imidazol numa taxa de fluxo de 0,5-1,0 ml/min (Wang et al, 2013 (Holmgren, Arne, 1985). Ruocco e cols.…”