• Title/Summary/Keyword: novel protein

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Purification and Properties of Novel Calcium-binding Proteins from Streptomyces coelicolor

  • Chang, Ji-Hun;Yoon, Soon-Sang;Lhee, Sang-Moon;Park, I-Ha;Jung, Do-Young;Park, Young-Sik;Yim, Jeong-Bin
    • Journal of Microbiology
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    • v.37 no.1
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    • pp.21-26
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    • 1999

  • Two novel calcium-binding proteins, named CAB-I and CAB-II, have been isolated from Streptomyces coelicolor. Purification of the calcium-binding proteins involved heat treatment, fractionation with ammonium sulfate, acid treatment, anion exchange and hydrophobic interaction column chromatography, FPLC gel filtration, and preparative isoelectric focusing. A chelex competitive assay and 45Ca autoradiography verified the calcium-binding ability of the proteins. The major band CAB-II has an apparent molecular weight of 26,000 determined by SDS-polyacrylamide gel electrophoresis and 340,000 determined by gel filtration. The isoelectric point of this molecule showed the acidic nature of the molecule. N-terminal amino acid sequence analysis shows homology to rat Ca2+/calmodulin-dependent protein kinase-II (CAB-II) and yeast phosphoprotein phosphatase (CAB-I).

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Novel sinIR promoter for Bacillus subtilis DB104 recombinant protein expression system

  • Ji-Su Jun;Min-Joo Kim;KwangWon Hong
    • Journal of Applied Biological Chemistry
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    • v.66
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    • pp.128-137
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    • 2023

  • Transcriptome analysis revealed that the sinR gene encoding a transition-state regulator of Bacillus pumilus, genetically close to B. subtilis, was expressed at high levels during growth. The sinR gene is the second gene of the sinIR operon consisting of three promoters and two structural genes in B. subtilis. This study used the sinIR promoter of B. subtilis DB104 to construct a recombinant protein expression system. First, the expression ability depending on the number of sinIR promoter was investigated using enhanced green fluorescent protein (eGFP). The expression level of eGFP was slightly higher when using two promoters (Psin2) than using original promoters. The Psin2 promoter was further engineered by modifying the repressor binding site and -35 and -10 regions. Shine-Dalgarno (SD) sequence of the sinI gene was modified to the consensus sequence. Finally, combining the engineered Psin2 promoter with the modified SD sequence increased the expression level of eGFP by about 13.4-fold over the original promoter. Our results suggest that the optimized sinIR promoter could be used as a novel tool for recombinant protein expression in B. subtilis.

  • https://doi.org/10.3839/jabc.2023.019 인용 PDF