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Influence of key residues on the heterologous extracellular production of fungal ribonuclease U2 in the yeast Pichia pastoris

Álvarez García, Elisa and García Ortega, Lucía and De los Ríos, Vivian and Gavilanes, José G. and Martínez del Pozo, Álvaro (2009) Influence of key residues on the heterologous extracellular production of fungal ribonuclease U2 in the yeast Pichia pastoris. Protein Expression and Purification, 65 . pp. 223-229. ISSN 1096-0279

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Abstract

Ribonuclease U2, secreted by the smut fungus Ustilago sphaerogena, is
a cyclizing ribonuclease that displays a rather unusual specificity within the
group of microbial extracellular RNases, best represented by RNase T1.
Superposition of the three-dimensional structures of RNases T1 and U2
suggests that the RNase U2 His 101 would be the residue equivalent to the
RNase T1 catalytically essential His 92. RNase U2 contains three disulfide
bridges but only two of them are conserved among the family of fungal
extracellular RNases. The non-conserved disulfide bond is established between
Cys residues 1 and 54. Mispairing of the disulfide network due to the presence
of two consecutive Cys residues (54 and 55) has been invoked to explain the
presence of wrongly folded RNase U2 species when produced in P. pastoris. In
order to study both hypotheses, the RNase U2 H101Q and C1/54S variants
have been produced, purified, and characterized. The results obtained support
the major conclusion that His 101 is required for proper protein folding when
secreted by the yeast P. pastoris. On the other hand, substitution of the first Cys
residue for Ser results in a mutant version which is more efficiently processed in
terms of a more complete removal of the yeast α-factor signal peptide. In
addition, it has been shown that elimination of the Cys 1-Cys 54 disulfide bridge
does not interfere with RNase U2 proper folding, generating a natively folded
but much less stable protein.

Item Type:Article
Uncontrolled Keywords:Microbial ribonucleases, RNase T1, Fungal ribonucleases, Disulfide
Subjects:Sciences > Chemistry > Biochemistry
ID Code:9140
Deposited On:08 Jun 2009 14:21
Last Modified:07 Sep 2010 08:30

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