Molecular Cloning And Protein Expression Of CDNAs Of Ndrg Family Genes Plus Generation Of Antibodies Specific To NDRG Proteins

The human ndrg2 (N-myc downstream regulated gene 2) cDNA was first cloned from normal human whole brain cDNA library in our lab in 2000. The full length of ndrg2 cDNA is 2,024 bp long. BLAST analysis verified that the discovered cDNA sequence represented a new gene (GenBank Accession AF 159092) located on human chromosome 14q11.2. ndrg2 gene consists of 16 exonsand 15 introns that encodes a 40 kDa protein with 357 amino acid residues. Preliminary data indicated that ndrg2 was expressed in multiple tissues, with heterogeneous expression in normal brain structures except glioma specimens lacking ndrg2 expression. Until now the reported four members comprise the ndrg family: ndrgl, ndrg2, ndrg3 and ndrg4, with ndrg4 possessing three isoforms: ndrg4-B, ndrg4-Bvar and ndrg4-H. The four ndrg family members share great homology in their composition of amino acid residues with differential expression in diverse tissues.Many factors might affect the expression of ndrgl gene. The expression of ndrgl is upregulated by homocystein, tunicamycin, DNA damaging agents, stressful stimuli (such as hypoxia and nickel toxication) and p53, and downregulated by androgen and the proto-oncogenes N-myc and c-myc. Existing evidences suggest that ndrgl and ndrg2 play active roles in cell proliferation and differentiation events, maintaining the balance of cell redox potential and suppressing metastasis of malignant tumor cells. As the structure and function of ndrg family genes remain largely unkown, it is very important to firstly clone and express cDNA sequences of ndrg family genes (ndrgl, 3 and 4) coupled with generation of specific antibodies against NDRG proteins. These will greatly facilitate subsequent comprehensive characterization of NDRG functions.Our study were therefore undertaken to address the above issues.1. cDNA cloning of ndrg family genes. Total RNA from human fetal brain tissue were extracted, then reverse transcribed into cDNA. Three pairs of primers were synthesized which target partial sequences in ndrgl, ndrg3 and ndrg4-B according to GenBank deposited sequences. PCR products were inserted into pMD18-T vector and sequenced. Sequences thus obtained were compared toGenBank database using BLAST program.2. The prokaryotic expression of ndrg family. The validated gene fragments were respectively inserted into pRSET-A, pGEX-4T-l and pPROEX-HTb expression vectors. Protein expression was induced by IPTG in E.coli. Bacterial strains that expressed highly soluble 6His-NDRGl in LB medium were obtained by transformation of pPROEX-HTb-ndrgl recombinant plasmid when analyzing their solubility.3. The secondary structural analysis of NDRG 1 fusion protein. The expressed fusion protein was purified by Ni-NTA agarose beads affinity chromatography. The secondary structure of purified 6His-NDRGl fusion protein was analyzed by circular dichroism. The results indicated that 6His-NDRGl was composed of a -Helix:23.6%,P-sheet:18.6%, Turn:25.7%, Random:32.0%. The outcome of analysis basically conforms to predicted data using computer programs.4. Polyclonal antibody preparation against NDRG family. Immunization of rabbits with fusion protein produced high titer polyclonal antibodies against NDRG1. The specific antibody was purified by absorbing antiserum with NDRG1 antigen immobilized on NC filters. Satisfactory results were obtained in immunohistochemical staining and Western blot using the generated antibodies. Due to the high homology in composition of amino acids residues in predicted ndrg family proteins, the antibody prepared against NDRG1 antigen failed to differentiate the four members of NDRG family. Thus an antibody against all NDRG family proteins was obtained.5. Polyclonal antibody preparation against the peptides of NDRG family proteins. We synthesized four short peptides as haptens that represent unique segments in protein sequences of NDRG1, NDRG2, NDRG3 and NDRG4respectively. These peptides conjugated with Keyhole limpet hemocyanin (KLH) were recently u...