Purification and characterization of Chromatium vinosum GroEL and GroES proteins overexpressed in Escherichia coli cells lacking the endogenous groESL operon.
Using an Escherichia coli strain (RF101) in which the endogenous chromosomal groESL operon was removed, we overexpressed the GroEL and GroES chaperonins cloned from the photosynthetic bacterium Chromatium vinosum. The identities of these proteins were confirmed by immunological and N-terminal sequence analyses. The native molecular masses of GroEL and GroES, as determined by size-exclusion chromatography, were 830 and 74 kDa, respectively. This suggests a tetradecameric structure for GroEL and a heptameric structure for GroES. C. vinosum GroEL catalyzed a K+-stimulated ATP hydrolysis with a specific activity at 25 degreesC of 50.2 +/- 3.8 nmol Pi released min-1 mg protein-1. GroEL ATPase was inhibited by GroES, reaching about 50% inhibition at a ratio GroES-7mer/GroEL-14mer of 1 in the presence of 10 mM KCl. The ATPase Vmax increased almost fivefold in the 25 to 65 degreesC temperature range; higher temperatures led to a rapid inactivation of this activity. The chaperone activity of the C. vinosum GroEL/GroES system was characterized by its effect on the refolding of guanidinium chloride-unfolded rhodanese. In the presence of ATP and GroES, C. vinosum GroEL assisted rhodanese refolding. The heterologous combination C. vinosum GroEL/E. coli GroES or E. coli GroEL/C. vinosum GroES was as effective as the homologous complexes. In summary, this strategy allowed the purification at high yields of fully functional, homogenous C. vinosum GroEL and GroES chaperonins from E. coli.
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Programa Multidisciplinario de Biología Experimental (CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, 2000, Argentina.
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D018835:Chaperonin 10 / Q000737:chemistry*; D018834:Chaperonin 60 / Q000737:chemistry*; D018833:Chaperonins / Q000235:genetics*; D002844:Chromatium / Q000737:chemistry*; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D007700:Kinetics; D009876:Operon / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry*; D017421:Sequence Analysis; D013696:Temperature; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; C097970:GroESL protein, Bacteria; D011994:Recombinant Proteins; D013884:Thiosulfate Sulfurtransferase; D000251:Adenosine Triphosphatases; D018833:Chaperonins