PCR-BASED CLONING OF THE FULL-LENGTH NEUROSPORA EUKARYOTIC INITIATION-FACTOR 5A CDNA - POLYHISTIDINE-TAGGING AND OVEREXPRESSION FOR PROTEINAFFINITY BINDING

Eukaryotic initiation factor 5A. (eIF-5A) is the only cellular protein known to contain a hypusine residue that is formed by transferring th e aminobutyl moiety from spermidine to a specific lysine residue, foll owed by hydroxylation at the aminobutyl group. A simple PCR-based stra tegy was developed to obtain a full-length cDNA of Neurospora crassa e IF-5A. The strategy consists of (i) the design of a pair of key primer s (21-mer) based on the highly conserved eIF-5A cDNA domains known in other species, (ii) PCR amplification of Neurospora cDNA using the two key primers to obtain the core sequence for the design of core primer s, and (iii) combined use of the key primers, core primers and the uni versal primers, T3 and T7, to amplify the target sequence in a Neurosp ora cDNA library. The longest cDNA obtained was cloned into pBlueScrip t phagemid, and sequence analysis indicated that it encodes a polypept ide of 163 amino acid residues with a codon usage preference character istic of abundant Neurospora genes. The Neurospora polypeptide showed 59 % and 67 % identity with human and yeast eIF-5A precursor protein r espectively. We subcloned the Neurospora eIF-5A cDNA into pQE-30, whic h introduces six adjacent histidine residues to the N-terminus of the recombinant protein. The resulting plasmid, pQTy21, was overexpressed in Escherichia coli, and the soluble polyhistidine-tagged protein was purified by metal chelation chromatography. We obtained about 60 mg of purified eIF-5A precursor from 1 litre of culture in a single step us ing a Ni(II)-nitrilotriacetic acid (NTA)-agarose column. The histidine -tagged eIF-5A precursor protein could be recognized by anti-Neurospor a crassa 21 kDa protein serum raised against wild-type eIF-5A precurso r and could serve as the substrate protein for deoxyhypusine synthase. Using the histidine-tagged recombinant protein and the Ni(II)-NTA-aga rose column, we constructed a protein affinity column and demonstrated an affinity binding between eIF-5A precursor and deoxyhypusine syntha se in the presence of NAD(+). One-step eIF-5A precursor affinity-colum n chromatography could lead to a 30-fold purification of deoxyhypusine synthase.