Efficient expression and purification of biologically active human cystatin proteins

Chauhan, Sakshi; Tomar, Raghuvir Singh

doi:10.1016/j.pep.2015.10.005

Cited by 6 publications

(5 citation statements)

References 26 publications

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“…For the purification of GST-tagged stefin B, the previously described protocol was followed. BL21 (DE3) cells transformed with the pGEX6P1 stefin B plasmid were grown in LB medium at 37 °C until the OD 600 reached 0.6.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…The stefin B gene was cloned and expressed as described previously. 22 For the cloning of stefin B, human stefin B was amplified from the pcDNA 3.1 stefin B vector and subcloned into the pGEX6P1 vector at EcoRI and XhoI restriction sites. Through PCR site-directed mutagenesis, point and truncation mutations were introduced into stefin B.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Cells were grown at 37 °C in LB medium (100 μg/mL ampicillin and 25 μg/mL chloramphenicol) until the OD 600 reached 0. For the purification of GST-tagged stefin B, the previously described protocol 22 was followed. BL21 (DE3) cells transformed with the pGEX6P1 stefin B plasmid were grown in LB medium at 37 °C until the OD 600 reached 0.6.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Despite many reports, regulation of such an irreversible process is not very well established in comparison to other epigenetic mechanisms. To understand the regulation of H3 clipping, we have identified stefin B and other cystatins as inhibitors of histone H3 protease . However, clipping sites and the mechanism of inhibition of H3 protease by stefin B are not known.…”

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confidence: 99%

“…To understand the regulation of H3 clipping, we have identified stefin B and other cystatins as inhibitors of histone H3 protease. 22 However, clipping sites and the mechanism of inhibition of H3 protease by stefin B are not known. In this study, we have identified three different highly dynamic clipping sites within the N-terminal tail region of histone H3.…”

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confidence: 99%

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Biochemical Analysis Reveals the Multifactorial Mechanism of Histone H3 Clipping by Chicken Liver Histone H3 Protease

2016

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Proteolytic clipping of histone H3 has been identified in many organisms. Despite several studies, the mechanism of clipping, the substrate specificity, and the significance of this poorly understood epigenetic mechanism are not clear. We have previously reported histone H3 specific proteolytic clipping and a protein inhibitor in chicken liver. However, the sites of clipping are still not known very well. In this study, we attempt to identify clipping sites in histone H3 and to determine the mechanism of inhibition by stefin B protein, a cysteine protease inhibitor. By employing site-directed mutagenesis and in vitro biochemical assays, we have identified three distinct clipping sites in recombinant human histone H3 and its variants (H3.1, H3.3, and H3t). However, post-translationally modified histones isolated from chicken liver and Saccharomyces cerevisiae wild-type cells showed different clipping patterns. Clipping of histone H3 N-terminal tail at three sites occurs in a sequential manner. We have further observed that clipping sites are regulated by the structure of the N-terminal tail as well as the globular domain of histone H3. We also have identified the QVVAG region of stefin B protein to be very crucial for inhibition of the protease activity. Altogether, our comprehensive biochemical studies have revealed three distinct clipping sites in histone H3 and their regulation by the structure of histone H3, histone modifications marks, and stefin B.

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Section: Materials and Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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