title: Thermo and pH stable ATP-independent chaperone activity of heat-inducible Hsp70 from Pennisetum glaucum.
abstract: Heat shock proteins (Hsps) are a class of molecular chaperones that play an essential role in preserving cellular functions under stressful conditions. The over production of recombinant proteins often causes cellular stress that results in aggregation/misfolding of proteins, which sometimes leads to the formation of inclusion bodies. Here we report the cloning and characterization of heat-inducible PgHsp70 from Pennisetum glaucum, a heat and drought tolerant plant that showed stability and chaperone activity at elevated temperatures. The predicted amino acid sequence of PgHsp70 revealed a high homology with Hsp70 from other plants, and the overall 3D structure homology modeling is similar to that of the constitutively expressed bovine cytosolic Heat Shock Cognate (HSC)-70. The purified recombinant protein had an apparent molecular mass of 70 kDa and displayed optimal chaperone activity at 50 degrees C, and pH 8.0. Under these conditions, the T(1/2) of PgHsp70 increased from 10 to 15 h in the presence of glycerol. The PgHsp70 exhibited a higher chaperone activity towards glutamate dehydrogenase than alcohol dehydrogenase. The expression of recombinant carbonic anhydrase (CA) in E. coli in a catalytically active soluble form rather than in inclusion bodies was made feasible by co-expression of PgHsp70. Circular dichroism (CD) studies of the recombinant PgHsp70 did not reveal any discernible changes in the alpha-helix content, with increase in temperature from 35 to 85 degrees C, thus suggesting a critical role of alpha-helix content in maintaining the chaperone activity.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: J L Uma Maheswar
authors.initials: JL
authors.lastname: Rao
authors.forename: Palakolanu Sudhakar
authors.initials: PS
authors.lastname: Reddy
authors.forename: Rabi N
authors.initials: RN
authors.lastname: Mishra
authors.forename: Dinesh
authors.initials: D
authors.lastname: Gupta
authors.forename: Dinkar
authors.initials: D
authors.lastname: Sahal
authors.forename: Narendra
authors.initials: N
authors.lastname: Tuteja
authors.forename: Sudhir K
authors.initials: SK
authors.lastname: Sopory
authors.forename: Malireddy K
authors.initials: MK
authors.lastname: Reddy
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D031782:Pennisetum / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: D018840:HSP70 Heat-Shock Proteins; D010940:Plant Proteins; D000426:Alcohol Dehydrogenase; D005969:Glutamate Dehydrogenase; D002256:Carbonic Anhydrases