title: Molecular cloning and expression of high GC-rich novel tumor suppressor gene HIC-1.
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)2SO4 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 °C. Full-length clone successfully expressed in BL21-Codon Plus-RP using 1 mM concentration of IPTG for 8 h at 37 °C gave prominent band of 74 kDa.
authors.affiliation: Biomolecular Science Centre, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Bld 20, 4110 Libra Drive, Orlando, FL, 32816, USA, sanjay.kumar@ucf.edu.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Kumar
mesh_terms: D001482:Base Composition; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D016147:Genes, Tumor Suppressor; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: C499808:HIC1 protein, human; D051741:Kruppel-Like Transcription Factors