Cloning, expression and physicochemical characterization of sugar cane hemoglobin.
Daniel H.A. Corrêab; Silvia L.F. Silvaa;,Carlos H. I. Ramosa#
aLaboratório Nacional de Luz Síncrotron, Campinas SP, Brazil; bInstituto de Biologia, UNICAMP, Campinas SP, Brazil.
#Caixa Postal 6192. CEP 13084-971. carlos.ramos@pesquisador.cnpq.br
Hemoglobin has been found in bacteria, plants, fungi, and animals. The first description of a hemoglobin from plant was made in late 30's and since then a large number of plant hemoglobins has been described. Hemoglobins were first identified in plants species which fix nitrogen via symbiosis with bacteria. It is likely that all plants have a hemoglobin gene and in addition to the symbiotic leghemoglobins known in legumes, there may be another gene or gene family in legumes encoding hemoglobins operative in nonsymbiotic plant tissues. Here we report the identification of a hemoglobin cDNA clone in the sugarcane expression sequence tag library SUCEST. To address issues of the sugarcane (SC) globin structure and stability, we have undertaken purification and spectral analysis of the sugarcane hemoglobin gene. The cDNA encoding the SC hemoglobin was cloned into a plasmid expression vector (pET3a) and overexpressed in Escherichia coli BL21(DE3)pLysS cells. Recombinant SC hemoglobin was purified to homogeneity and the examination of expression yield and protein profile purification was performed by SDS-PAGE. The purified apoSC hemoglobin was soluble, showing stable a-helical conformation as seen by its characteristic Circular Dichroism spectra; it was able to bind heme as seen by its spectra at the Soret band in the 400 nm. The stabilities of both apo and holo SC hemoglobin were investigated by heat-induced unfolding monitored by CD at 222 nm showing that the apo form is more stable than the holo form. The acid-induced unfolding profile of the apoSC hemoglobin was monitored by both far-UV CD at 222 nm and by intrinsic emission fluorescence showing that, different from other well studied globins, apoSC remained folded at acidic pH.
Supported by FAPESP and MCT/CNPq
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