Expression in Escherichia coli, purification and functional activity of recombinant human chaperonin 10.
We have recently reported the cloning of a cDNA coding for a stress inducible human chaperonin 10. The protein was shown to possess 100% identity with the bovine homologue and a single amino acid replacement (glycine to serine at position 52) compared to rat chaperonin 10. Here we report the heterologous expression of human chaperonin 10 in Escherichia coli, its purification and its functional characterization. The recombinant protein was purified to homogeneity as judged by different analytical techniques, and mass spectrometry analysis showed a MW of 10,801 Da in agreement with the predicted sequence. This molecular weight accounts for a protein which is not modified post-translationally. In fact, natural rat chaperonin 10 has been shown to be acetylated at the N-terminus, a feature suggested to be important for targeting and functional activity. Here we show that recombinant human chaperonin 10 is fully active in assisting the chaperonin 60 GroEL in the refolding of denatured yeast enolase, thereby showing that, at least in the present system, post-translational acetylation is not necessary for its activity.
Legname
G
G
Italfarmaco Research Center, Milano, Italy.
Fossati
G
G
Gromo
G
G
Monzini
N
N
Marcucci
F
F
Modena
D
D
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D018835:Chaperonin 10 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D018834:Chaperonin 60 / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D018835:Chaperonin 10; D018834:Chaperonin 60; D017931:DNA Primers; D011994:Recombinant Proteins