Human ribosomal protein S13: cloning, expression, refolding, and structural stability.
The cDNA of human ribosomal protein S13 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in Escherichia coli cells. Protein accumulated in the form of inclusion bodies was isolated, purified, and refolded by dialysis. The recombinant protein was immunologically reactive, interacting with antiserum against native rpS13. The secondary structure content of the refolded protein was analyzed by means of CD spectroscopy. It was found that 43+/-5% of amino acids sequence of the protein form alpha-helices and 11+/-3% are placed in beta-strands that coincides with theoretical predictions. The beta-strands seem to be located in the extension regions of the rpS13 and do not have homologuous regions in the structure of rpS15 from Thermus thermophilus, which is a prokaryotic homolog of rpS13. The protein structure is stable at a pH range from 4.0 to 8.0 and at low concentrations of urea (up to 3 M).
Malygin
Alexey
A
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva, 8, Novosibirsk, 630090, Russia.
Parakhnevitch
Natalia
N
Karpova
Galina
G
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000235:genetics
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; C047013:RPS13 protein, human; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C512838:rpsM protein, E coli