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Palacios F.,Seccion Bioquimica Biologia Molecular | Cota G.,Seccion Bioquimica Biologia Molecular | Horjales S.,Seccion Bioquimica Biologia Molecular | Lima A.,Institute Pasteur Of Montevideo | And 3 more authors.
Biotechnology Journal | Year: 2010

'Conformational diseases' are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1G93A mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue-specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody-based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSODG93A rats provided evidence about heterodimers rSOD-hSODG93A formation in both extracts. Moreover, when characterized by 2-DE and MALDI-TOF/TOF MS, the extracted hSODG93A showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post-translational modifications of hSOD1 produced in various tissues. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

Ramon A.,Seccion Bioquimica Biologia Molecular | Marin M.,Seccion Bioquimica Biologia Molecular
Biotechnology Journal | Year: 2011

Membrane proteins play key roles in diverse cellular functions and have become the target for a large number of pharmacological drugs. Despite representing about 20-30% of cellular proteins, their characterization is long overdue since they are difficult to handle, to purify from their natural source or to obtain as recombinant proteins. Pichia pastoris is a methylotrophic yeast species increasingly used as a host for heterologous protein expression for both research and industrial purposes. Over the past few years many efforts have allowed important advances in the development of this expression system for the expression and production of membrane proteins. The most recent achievements in improving yield and proper folding of integral membrane proteins are summarized in this review. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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