PCR-based cloning of the full-length Neurospora eukaryotic initiation factor 5A cDNA: polyhistidine-tagging and overexpression for protein affinity binding.
Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain a hypusine residue that is formed by transferring the aminobutyl moiety from spermidine to a specific lysine residue, followed by hydroxylation at the aminobutyl group. A simple PCR-based strategy was developed to obtain a full-length cDNA of Neurospora crassa eIF-5A. The strategy consists of (i) the design of a pair of key primers (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 primers, and (iii) combined use of the key primers, core primers and the universal primers, T3 and T7, to amplify the target sequence in a Neurospora cDNA library. The longest cDNA obtained was cloned into pBlueScript phagemid, and sequence analysis indicated that it encodes a polypeptide of 163 amino acid residues with a codon usage preference characteristic of abundant Neurospora genes. The Neurospora polypeptide showed 59% and 67% identity with human and yeast eIF-5A precursor protein respectively. We subcloned the Neurospora eIF-5A cDNA into pQE-30, which 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 using a Ni(II)-nitrilotriacetic acid (NTA)-agarose column. The histidine-tagged eIF-5A precursor protein could be recognized by anti-Neurospora crassa 21 kDa protein serum raised against wild-type eIF-5A precursor and could serve as the substrate protein for deoxyhypusine synthase. Using the histidine-tagged recombinant protein and the Ni(II)-NTA-agarose column, we constructed a protein affinity column and demonstrated an affinity binding between eIF-5A precursor and deoxyhypusine synthase in the presence of NAD+. One-step eIF-5A precursor affinity-column chromatography could lead to a 30-fold purification of deoxyhypusine synthase.
Tao
Y
Y
Department of Chemistry, Rutgers University, Piscataway, NJ 08855-0939.
Chen
K Y
KY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009491:Neurospora / Q000235:genetics* / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification* / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011498:Protein Precursors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D000097574:Eukaryotic Translation Initiation Factor 5A
D018076:DNA, Complementary; D006639:Histidine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D010455:Peptides; D011498:Protein Precursors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C033223:polyhistidine; C063507:deoxyhypusine synthase