title: Expression and characterization of the Arabidopsis thaliana 11S globulin family.
abstract: The 11S globulins are the principal seed storage proteins in a variety of major crop species, including members of the legume and mustard families. They are targets for protein engineering studies attempting to alter the physicochemical properties of seed protein extracts (e.g. soybean) and to improve the nutritional quality of important agricultural crops. A key factor that has limited the success of this approach to date is insufficient accumulation of the engineered protein variants in vivo due to their improper folding and/or reduced stability, compared to the native protein. We have developed the Arabidopsis thaliana 11S proglobulins as a model system to enable studies exploring the factors underlying structural stability in this family of proteins. Yields of 1.5-4 mg/L were achieved for the three A. thaliana 11S proglobulins expressed in the Origami Escherichia coli cell line in super broth media at 20°C for 16 h and purified via immobilized-metal affinity chromatography. We also demonstrate that differential scanning fluorimetry is an effective and accessible technique to facilitate the screening of variants to enable the successful engineering of 11S seed storage proteins. The relative in vitro stability of the A. thaliana 11S proglobulins (proAtCRU1>proAtCRU3>proAtCRU2) is consistent between chemical and thermal denaturation studies.
authors: Department of Biology, Carleton University, Ottawa K1S 5B6, Canada. Electronic address: allisonjaworski@cmail.ca.
authors: Allison F
authors: AF
authors: Jaworski
authors: Department of Biology, Carleton University, Ottawa K1S 5B6, Canada; Department of Health Sciences, Carleton University, Ottawa K1S 5B6, Canada. Electronic address: susan_aitken@carleton.ca.
authors: Susan M
authors: SM
authors: Aitken
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005470:Fluorometry / Q000379:methods; D005916:Globulins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D015202:Protein Engineering; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D055314:Seed Storage Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D005916:Globulins; D020033:Protein Isoforms; D011498:Protein Precursors; D011994:Recombinant Proteins; D055314:Seed Storage Proteins
keywords: Arabidopsis thaliana; Differential scanning fluorimetry; Procruciferin; Protein engineering; Seed storage protein