title: A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies.
abstract: The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation.
authors: Biotechnology Department, IPEN-CNEN, SP, Brazil.
authors: Larissa M
authors: LM
authors: Pereira
authors: Biotechnology Department, IPEN-CNEN, SP, Brazil.
authors: Luana R
authors: LR
authors: Silva
authors: Biotechnology Department, IPEN-CNEN, SP, Brazil.
authors: Joseane F
authors: JF
authors: Alves
authors: Biochemistry Department, IQUSP, Brazil.
authors: Nélida
authors: N
authors: Marin
authors: Biotechnology Department, IPEN-CNEN, SP, Brazil.
authors: Flavio Sousa
authors: FS
authors: Silva
authors: Biotechnology Department, IPEN-CNEN, SP, Brazil.
authors: Ligia
authors: L
authors: Morganti
authors: Molecular Gynaecology Laboratory, UNIFESP, SP, Brazil.
authors: Ismael D C G
authors: ID
authors: Silva
authors: Biotechnology Department, IPEN-CNEN, SP, Brazil; Molecular Gynaecology Laboratory, UNIFESP, SP, Brazil. Electronic address: reginaffonso@yahoo.com.br.
authors: Regina
authors: R
authors: Affonso
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry
chemical_list: C000630686:RPL10 protein, human; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D025521:Tumor Suppressor Proteins; D015032:Zinc
keywords: Binding zinc ions; Correct folding; Inclusion bodies; Soluble protein