title: A general method of protein purification for recombinant unstructured non-acidic proteins.
abstract: Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.
authors.affiliation: Departmento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico. campos@ibt.unam.mx
authors.forename: Francisco
authors.initials: F
authors.lastname: Campos
authors.forename: Gabriel
authors.initials: G
authors.lastname: Guillén
authors.forename: José L
authors.initials: JL
authors.lastname: Reyes
authors.forename: Alejandra A
authors.initials: AA
authors.lastname: Covarrubias
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000302:isolation & purification*; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005615:Freezing; D007526:Isoelectric Point; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification*; D014238:Trichloroacetic Acid / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D014238:Trichloroacetic Acid