Overexpression and Large-Scale Purification of Recombinant Hamster Polymorphic ArylamineN-Acetyltransferase as a Dihydrofolate Reductase Fusion Protein

Journal of Biological Chemistry

Tikh

Horta

et al. 2007

Self Cite

Hint1 is a homodimeric protein and member of the ubiquitous HIT superfamily. Hint1 catalyzes the hydrolysis of purine phosphoramidates and lysyl-adenylate generated by lysyl-tRNA synthetase (LysRS). To determine the importance of homodimerization on the biological and catalytic activity of Hint1, the dimer interface of human Hint1 (hHint1) was destabilized by replacement of Val 97 of hHint1 with Asp, Glu, or Arg. The mutants were shown to exist as monomers in solution by a combination of size exclusion chromatograph, static light scattering, and chemically induced dimerization studies. Circular dichroism studies revealed little difference between the stability of the V97D, V97E, and wild-type hHint1. Relative to wild-type and the V97E mutant, however, significant perturbation of the V97D mutant structure was observed. hHint1 was shown to prefer 3-indolepropionic acyl-adenylate (AIPA) over tryptamine adenosine phosphoramidate monoester (TpAd). Wild-type hHint1 was found to be 277-and 1000-fold more efficient (k cat /K m values) than the V97E and V97D mutants, respectively. Adenylation of wildtype, V97D, and V97E hHint1 by human LysRS was shown to correlate with the mutant k cat /K m values using 3-indolepropionic acyl-adenylate as a substrate, but not tryptamine adenosine phosphoramidate monoester. Significant perturbations of the active site residues were not detected by molecular dynamics simulations of the hHint1s. Taken together, these results demonstrate that for hHint1; 1) the efficiency (k cat / K m ) of acylated AMP hydrolysis, but not maximal catalytic turnover (k cat ), is dependent on homodimerization and 2) the hydrolysis of lysyl-AMP generated by LysRS is not dependent on homodimerization if the monomer structure is similar to the wild-type structure.Histidine triad nucleotide-binding proteins (Hint) 2 are members of the histidine triad (HIT) protein superfamily of nucleotidyltransferases and hydrolyases (1). HIT proteins are named for the conserved nucleotide binding motif containing the sequence His-X-His-X-His-XX, in which X is a hydrophobic amino acid. The HIT proteins have been classified into three subfamilies according to their enzymatic function, sequence composition, and structural similarity; the Hint branch, the fragile histidine triad (Fhit) branch, and the galactose-1-phosphate uridyltransferase (GalT) branch (1).The Hint branch is the most ancient and can be found in Archaea, Bacteria, and Eukaryotae. Hints are homodimeric proteins that have been found to be purine phosphoramidases (2-5). Recently, we have demonstrated that lysyl-adenylate (lysyl-AMP) generated by bacterial and human lysyl-tRNA synthetases (LysRS) are also substrates for the respective bacterial and human Hints (6). Escherichia coli hinT knock-out mutants exhibited salt-dependent cell growth, whereas Hint1 knock-out mice have an increased susceptibility to 7, 12-dimethylbenz[a]anthracene (DMBA)-induced ovarian and mammary tumors by the carcinogen DMBA and increased incidence of spontaneous tumors (2,7,8). In addition,...

Section: Methodsmentioning

confidence: 99%

“…Purification of wild-type fusion protein was published. Cell growth and cell lysate extractions were as previously described (26). The same purification procedure for each mutant was employed.…”

Section: Methodsmentioning

confidence: 99%

Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate

Journal of Biological Chemistry

Tikh

Horta

et al. 2007

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“…Plasmids pB432 and pB433 were obtained by cloning HindIII-EcoRI fragments containing wild-type and H101A mutated alleles of hinT from plasmids pB429 and pB431 into plasmid pACYC184. The expression construct pPH70D contains the E. coli dihydrofolate reductase (DHFR) gene followed by a thrombin cleavage site and the protein of interest (30). The fusion proteins are purified with a methotrexate affinity chromatography followed by cleavage with human thrombin to release the native protein.…”

Section: Methodsmentioning

confidence: 99%

“…Expression and Purification of Recombinant Proteins-The cell growth and cell lysate extraction were described previously except that 0.5 mM isopropyl-␤-D-thiogalactopyranoside was used for induction (30). Bacterial or human Hint-DHFR fusion proteins were purified by methotrexate-agarose (Sigma) using a 12.5-ml column, washed with 40 column volumes of buffer A, 60 column volumes of buffer A with 1 M NaCl, and followed by folate elution with 5 mM folate, 32 mM Tris, pH 9.0, 1 mM EDTA, 1 mM DTT, and 0.01 mM phenylmethylsulfonyl fluoride.…”

Section: Methodsmentioning

confidence: 99%

31P NMR and Genetic Analysis Establish hinT as the Only Escherchia coli Purine Nucleoside Phosphoramidase and as Essential for Growth under High Salt Conditions

Journal of Biological Chemistry

Bieganowski

Shilinski

et al. 2005

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Eukaryotic cells encode AMP-lysine (AMP-N-⑀-(N-␣acetyl lysine methyl ester) 5-phosphoramidate) hydrolases related to the rabbit histidine triad nucleotidebinding protein 1 (Hint1) sequence. Bacterial and archaeal cells have Hint homologs annotated in a variety of ways, but the enzymes have not been characterized, nor have phenotypes been described due to loss of enzymatic activity. We developed a quantitative 31 P NMR assay to determine whether Escherichia coli possesses an adenosine phosphoramidase activity. Indeed, soluble lysates prepared from wild-type laboratory E. coli exhibited activity on the model substrate adenosine 5-monophosphoramidate (AMP-NH 2 ). The E. coli Hint homolog, which had been comprehensively designated ycfF and is here named hinT, was cloned, overexpressed, purified, and characterized with respect to purine nucleoside phosphoramidate substrates. Bacterial hinT was several times more active than human or rabbit Hint1 on five model substrates. In addition, bacterial and mammalian enzymes preferred guanosine versus adenosine phosphoramidates as substrates. Analysis of the lysates from a constructed hinT knock-out strain of E. coli demonstrated that all of the cellular purine nucleoside phosphoramidase activity is due to hinT. Physiological analysis of this mutant revealed that the loss of hinT results in failure to grow in media containing 0.75 M KCl, 0.9 M NaCl, 0.5 M NaOAc, or 10 mM MnCl 2 . Thus, cation-resistant bacterial cell growth may be dependent on the hydrolysis of adenylylated and/or guanylylated phosphoramidate substrates by hinT.

“…The construction of the expression plasmid pPH70D containing dihydrofolate reductase (DHFR) and the FLAG peptide was described previously [31]. Cloning vector pSTBlue-1 was obtained from Novagen.…”

Section: Methodsmentioning

confidence: 99%

Expression, purification and characterization of recombinant mouse translation initiation factor eIF4E as a dihydrofolate reductase (DHFR) fusion protein

Ghosh

Cheng

Protein Expression and Purification

et al. 2008

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One of the earliest steps in translation initiation is recognition of the mRNA cap structure (m7GpppX) by the initiation factor eIF4E. Studies of interactions between purified eIF4E and its binding partners provide important information for understanding mechanisms underlying translational control in normal and cancer cells. Numerous impediments of the available methods used for eIF4E purification led us to develop a novel methodology for obtaining fractions of eIF4E free from undesired byproducts. Herein we report methods for bacterial expression of eIF4E tagged with mutant dihydrofolate reductase (DHFR) followed by isolation and purification of the DHFR-eIF4E protein by using affinity and anion-exchange chromatography. Fluorescence quenching experiments indicated the cap analogue, 7MeGTP, bound to DHFR-eIF4E and eIF4E with a dissociation constant (K d ) of 6±5 and 10±3 nM, respectively. Recombinant eIF4E and DHFR-eIF4E were both shown to significantly enhance in vitro translation in dose dependent manner by 75% at 0.5 uM. Nevertheless increased concentrations of eIF4E and DHFR-eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 uM of 60% and 90%, respectively. Thus, we have demonstrated that we have developed an expression system for fully functional recombinant eIF4E. We have also shown that the fusion protein DHFR-eIF4E is functional and thus may be useful for cell based affinity tag studies with fluorescently labeled trimethoprim analogs.