Recombinant expression of C-terminal domain of Rif1 and its solubilization with sarkosyl

Ghadiri, Hamed; Alavi, Sana; Dabirmanesh, Bahareh; Khajeh, Khosro

Recombinant expression of C-terminal domain of Rif1 and its solubilization with sarkosyl

Document Type : Original Research

Authors

Hamed Ghadiri

Sana Alavi

Bahareh Dabirmanesh

Khosro Khajeh

Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-175, Iran

Abstract

The eukaryotic genome contains several replication Origins. Studies showed that the phenomenon and order of the origin activation is in a a particular discipline, called the “Replication Timing". Recent studies show that many factors are involved in regulating the timing of the replication process. One of the most important factors amongst them is the Rap1 interacting Factor 1 (Rif1) protein, which plays a key role in regulating the replication schedule in yeast and more advanced eukaryotes. Structure of this protein is mostly irregular and these properties prevent Rif1 from being expressed in a stable manner and makes it difficult to study.

The aim of the present study was to investigate the expression of recombinant C-terminal domain of mouse-Rif1(muRif1-CTD) protein in solution. For this purpose, the muRif1-CTD gene was extracted from eukaryotic constructs containing the complete Rif1 gene by PCR and was inserted into the pPAL7 expression vector comprising the Profinity eXact tag. Protein solubilization was carried out using different detergents and then detergent removal was performed by dialysis. In order to ensure that the soluble protein is active, the interaction analysis of the Rif1 protein with the G4 structures (previously reported to bind Rif1) was investigated using the gel shift assay. The results of this study showed the use of detergent for Rif1 solubilization without affecting its purification steps. But in the case of this protein, if the detergent is removed completely, it will not remain soluble.

Keywords

Rif1 protein

sarcosyl

Solubilization

replication timing

expression in prokaryotic systems

20.1001.1.23222115.1399.12.1.6.8

Subjects

Molecular biotechnology

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