Title

Molecular Cloning and Expression of High GC-Rich Novel Tumor Suppressor Gene HIC-1

Authors

Authors

S. Kumar

Comments

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Abbreviated Journal Title

Mol. Biotechnol.

Keywords

HIC-1; GC-rich; Cloning; Protein expression; Tumor suppressor gene; 17P ALLELIC LOSS; ESCHERICHIA-COLI; P53; PCR; DNA; HYPERMETHYLATION; HETEROZYGOSITY; IDENTIFICATION; AMPLIFICATION; PROTEINS; Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology

Abstract

Hypermethylated in Cancer-1 (HIC-1) is a novel tumor suppressor plays crucial role in tumor formation through loss of function by hypermethylation. HIC-1 is known as transcriptional factor whereas little known about its structure and function. Requirement felt to clone and express full coding protein and reveal various domains and binding pattern onto promoters conducting biophysical studies which lack in current scenario. Production of sufficient amounts of protein is frequent bottleneck in structural biology. Cloning full-length HIC-1 with > 73 % GC content poses a daunting task with sequencing and expression adds more to the challenge. We describe the methodology for specific amplification, cloning, sequencing, and expression of HIC-1 in E. coli. Standardization using 1.5 U pfu polymerase in (NH4)(2)SO4 containing buffer gave specific amplification with 10 % DMSO and 1.5 mM MgCl2. Sequencing achieved using base analog 7-de aza dGTP (0.2 mM) or denaturant like DMSO (10 %) or betaine (1 M). Expression using strains of E. coli induced by different concentrations of IPTG (0.5-5.0 mM) for time points of 4, 8, 16, 20, and 24 h at different temperatures 25, 30, and 37 A degrees C. Full-length clone successfully expressed in BL21-Codon Plus-RP using 1 mM concentration of IPTG for 8 h at 37 A degrees C gave prominent band of 74 kDa.

Journal Title

Molecular Biotechnology

Volume

56

Issue/Number

11

Publication Date

1-1-2014

Document Type

Article

Language

English

First Page

1040

Last Page

1048

WOS Identifier

WOS:000342447800009

ISSN

1073-6085

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