Cloning, expression, purification and characterization of sorghum Hsp110 protein to understanding the protein disaggregation system in plants

Author(s)

Juliana Crotti Franco

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Química
• Defense date: 2018-03-22

Examining board members

Carlos Henrique Inacio Ramos; Fábio Cesar Gozzo; Jörg Kobarg

• Advisor: Carlos Henrique Inacio Ramos

Abstract

The Hsp100 chaperone, which is present from bacteria to plants, but is not found in metazoa, has the important function of disaggregase, aiding proteins to be recovered and refolded. Recent studies have suggested that in animal cells, the co-chaperone Hsp110 cooperates with Hsp70 in an efficient disaggregation system. As plants express both Hsp100 and Hsp110, we wondered whether Hsp110 could be part of an additional disaggregation system in this organism (sorghum in this case), since plants face more severe stressing conditions than animals. The hsp110 gene was identified in sorghum and the expression pET system was transformed in E. coli cells and the protein purified using nickel affinity chromatography followed by molecular exclusion chromatography. A combination of spectroscopic and hydrodynamic techniques was used to characterize protein conformation, while experiments involving protection against aggregation and assays of refolding and disaggregation of model substrates were used for the functional characterization. The protein was expressed soluble, and after purification it was found that it was produced folded (about 40 % of its polypeptide chain is in ? helix form) and stable, maintaining secondary structure up to 48 °C or in approximately 2 mole L-1 of urea. Hydrodynamic measurements indicated that the protein is an elongated monomer with a molecular mass of 98 kDa (expected value from sequence is 95.23 kDa). Hsp110 was also functional since it showed chaperone activity and was able, together with other proteins of the Hsp70 system, to refold and disaggregate a tested model substrate. These results support the hypothesis that plants have more than one system capable of acting in the disaggregation of proteins increasing their power to tolerate the different types of stress to which they are exposed (AU)

• FAPESP's process: 16/02137-1 - Cloning, expression, purification and characterization of sorghum Hsp110 protein aimed at understanding of protein disaggregation system in plants
• Grantee: Juliana Crotti Franco
• Support Opportunities: Scholarships in Brazil - Master