Molecular Cloning, Expression, and Purification of Nuclear Inclusion A Protease from Tobacco Vein Mottling Virus

Hwang, Dong Soo; Kim, Do-Hyung; Kang, Byoung Heon; Song, Byeong Doo; Choi, Kwan Yong

doi:10.1007/s10059-000-0148-8

Cited by 8 publications

(2 citation statements)

References 38 publications

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“…Thus, to effectively model rapid cellular processes, such as the import dynamics of histones, improvements with regard to cleavage kinetics will need to be made. In this regard, the downside to a viral protease's stringent specificity is its lower catalytic turnover, although a reported turnover rate of 11 s for TVMV suggests a significant increase in release kinetics is still achievable (Hwang et al, 2000;Nallamsetty et al, 2004;Sun et al, 2010). Despite these drawbacks, the ability to cytosolically tether H3.1 and H4 allowed us to investigate the nucleo-cytoplasmic divide at a level that has previously not been possible, bypassing the requirement for biochemical analysis, and enabling the observation of core histone incorporation into chromatin at actively replicating domains in living cells.…”

Section: Design Principles For the Rapid-release Approachmentioning

confidence: 99%

Evidence for the nuclear import of histones H3.1 and H4 as monomers

2018

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Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether‐and‐release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin‐alpha proteins (predominantly IPO4), but not to histone‐specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.

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Section: Design Principles For the Rapid-release Approachmentioning

confidence: 99%

Evidence for the nuclear import of histones H3.1 and H4 as monomers

2018

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“…Although TEV protease is by far the best studied and most widely used potyviral protease, several others have also been characterized to varying degrees. These include the TVMV protease [73,79], the plum pox virus protease [80], and the turnip mosaic virus protease [81,82]. None of these enzymes offer any compelling advantages over TEV protease, however.…”

Section: Tev Proteasementioning

confidence: 99%

An overview of enzymatic reagents for the removal of affinity tags

Waugh

2011

Protein Expression and Purification

303

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Although they are often exploited to facilitate the expression and purification of recombinant proteins, every affinity tag, whether large or small, has the potential to interfere with the structure and function of its fusion partner. For this reason, reliable methods for removing affinity tags are needed. Only enzymes have the requisite specificity to be generally useful reagents for this purpose. In this review, the advantages and disadvantages of some commonly used endo- and exoproteases are discussed in light of the latest information.

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Structural determinants of tobacco vein mottling virus protease substrate specificity

et al. 2010

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Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (~20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by~10-fold, an analogous truncation mutant of TVMV protease is significantly more active.Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1 0 position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k cat and K m for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

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Molecular Cloning, Expression, and Purification of Nuclear Inclusion A Protease from Tobacco Vein Mottling Virus

Cited by 8 publications

References 38 publications

Evidence for the nuclear import of histones H3.1 and H4 as monomers

Evidence for the nuclear import of histones H3.1 and H4 as monomers

An overview of enzymatic reagents for the removal of affinity tags

Structural determinants of tobacco vein mottling virus protease substrate specificity

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