Cloning, expression, characterization, and crystallization of a glutaminyl cyclase from human bone marrow: a single zinc metalloenzyme.
Glutaminyl cyclase (QC) catalyzes the N-terminal pyroglutamate formation of numerous hormones and peptides from their glutaminyl precursor. Pyroglutamate is a posttranslational or cotranslational modification important in many physiological and pathological processes. Here, we present the cloning of a QC cDNA from human bone marrow cDNA library. The protein was expressed in Escherichia coli system with the yields higher than approximately 10 mg/L bacterial culture, using a thioredoxin-tagged expression vector with several modifications. Based on high histidine content ( approximately 5%) of the protein, a convenient purification step by Ni-affinity chromatography was designed, leading to near homogeneity of the purified human QC. The identity of the recombinant human QC was confirmed by mass spectrometry and circular dichroism spectroscopy. The enzyme was active on both synthetic and physiological substrates, and the activity could be inhibited by several imidazole, triazole, and tetrazole derivatives. An atomic absorption analysis demonstrated that human QC contains one zinc ion per protein molecule. We also obtained the human QC crystals, which belong to cubic, tetragonal, and rhombohedral forms. Our works are useful to acquire new insights into human and animal QCs, particularly for future structural analysis and inhibitor designs.
Huang
Kai-Fa
KF
Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan.
Liu
Yi-Liang
YL
Wang
Andrew H-J
AH
D019881:Aminoacyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D001853:Bone Marrow / Q000378:metabolism*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D049308:Dioxygenases; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli; D015723:Gene Library; D006801:Humans; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000037:antagonists & inhibitors*; D008970:Molecular Weight; D013379:Substrate Specificity; D017967:Zinc Compounds / Q000037:antagonists & inhibitors
D004791:Enzyme Inhibitors; D008667:Metalloproteins; D017967:Zinc Compounds; D049308:Dioxygenases; C083734:aci-reductone oxidase (CO-forming); D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase