Cloning and expression of truncated CTCF
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Date
2005-03
Authors
Ngan, Chot San
Journal Title
Journal ISSN
Volume Title
Publisher
Pusat Pengajian Sains Kesihatan, Universiti Sains Malaysia
Abstract
The CTCF gene maps on chromosome 16 at 16q21- q22.3. It encodes a transcriptional
factor protein CTCF called zinc finger protein. CTCF has a number of functions in the
cell including controls of cell proliferation by having interactions with other proteins.
There are several proteins associated with CTCF. Among them are proteins involved in
transcriptional and cell proliferation control, RNA processing, signal transduction,
nucleosome components and tumor suppressors.
CTCF consists of N-tenninal domain, zinc finger domain and C-terminal domain.
Extensive research have been done to elucidate the important regions that might play
important role in both DNA binding and protein-protein interactions. Previous studies has
shown there was a region in the C-domain which has direct interaction with large subunit
of RNA polymerase IT and controlling various cellular process.
In this study, a truncated CTCF from the C-tenninal domain was produced by polymerase
chain reaction (PCR). The amplified products was then subcloned into the intermediate
cloning vector, pTOP02.1 and transformed into E. coli strain DH5a. The insert was then
cut with respective restriction enzyme to obtain the truncated region and further ligated
into pET16b expression vector for its expression in E. coli strain BL21 (DE3). Expressed
protein was separated using SDS-PAGE followed by Western Blotting using a-Histag
monoclonal antibody. The truncated CTCF protein was detected to migrate at the size of
approximately 27kDa despite its theoretical size of 4kDa. This migration was expected
and due to anomalous conformational changes during migration.
Description
Keywords
chromosome