Tri-cistronic cloning, overexpression and purification of human Rad9, Rad1, Hus1 protein complex

Singh, Vinay Kumar; Nurmohamed, Salima; Davey, Scott; Jia, Zongchao

doi:10.1016/j.pep.2007.03.011

Cited by 4 publications

(2 citation statements)

References 45 publications

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“…The solution behavior as well as the stoichiometry of the σ factor/anti-σ factor proteins were also examined by DLS [10, 11]. DLS measures the translational diffusion coefficients (D T ) of proteins by monitoring the scattered light produced when a monochromatic light is directed at a protein in solution.…”

Section: Resultsmentioning

confidence: 99%

“…The first involves cloning of target genes in a bi-cistronic vector, where genes are present in the same order as in an operon and the expression of the recombinant protein is controlled by a single promoter. A drawback of this approach is that the expression of second gene is usually lower, resulting in a sub-stoichiometric arrangement of the resultant complex [10, 17]. The second strategy involves co-transformation wherein the interacting partners are cloned in two different plasmids with different selection markers [18, 19].…”

Section: Discussionmentioning

confidence: 99%

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Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis σ/anti-σ factor protein complexes

Thakur¹,

Kumar²,

Shukla³

et al. 2010

Protein Expression and Purification

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The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the σ factor/anti-σ factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of σ factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis σ/anti-σ factor protein complexes

Thakur¹,

Kumar²,

Shukla³

et al. 2010

Protein Expression and Purification

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Using Affinity Pulldown Assays to Study Protein–Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9–RAD1–HUS1 (9-1-1) Complex

McDonald

Wang

Johnson

et al. 2023

Methods in Molecular Biology

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Mechanisms of Dealing with DNA Damage-Induced Replication Problems

Budzowska

Kanaar

2008

Cell Biochem Biophys

113

120

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During every S phase, cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. Given the large size of mammalian genomes and the required precision of DNA replication, genome duplication requires highly fine-tuned corrective and quality control processes. A major threat to the accuracy and efficiency of DNA synthesis is the presence of DNA lesions, caused by both endogenous and exogenous damaging agents. Replicative DNA polymerases, which carry out the bulk of DNA synthesis, evolved to do their job extremely precisely and efficiently. However, they are unable to use damaged DNA as a template and, consequently, are stopped at most DNA lesions. Failure to restart such stalled replication forks can result in major chromosomal aberrations and lead to cell dysfunction or death. Therefore, a well-coordinated response to replication perturbation is essential for cell survival and fitness. Here we review how this response involves activating checkpoint signaling and the use of specialized pathways promoting replication restart. Checkpoint signaling adjusts cell cycle progression to the emergency situation and thus gives cells more time to deal with the damage. Replication restart is mediated by two pathways. Homologous recombination uses homologous DNA sequence to repair or bypass the lesion and is therefore mainly error free. Error-prone translesion synthesis employs specialized, low fidelity polymerases to bypass the damage.

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Tri-cistronic cloning, overexpression and purification of human Rad9, Rad1, Hus1 protein complex

Cited by 4 publications

References 45 publications

Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis σ/anti-σ factor protein complexes

Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis σ/anti-σ factor protein complexes

Using Affinity Pulldown Assays to Study Protein–Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9–RAD1–HUS1 (9-1-1) Complex

Mechanisms of Dealing with DNA Damage-Induced Replication Problems

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