Molecular Cloning and Expression of High GC-Rich Novel Tumor Suppressor Gene HIC-1
Abbreviated Journal Title
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
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.
"Molecular Cloning and Expression of High GC-Rich Novel Tumor Suppressor Gene HIC-1" (2014). Faculty Bibliography 2010s. 5608.