Expression and purification of human ribosomal proteins S3, S5, S10, S19, and S26.
The cDNAs for the human ribosomal proteins S3, S5, S10, S19, and S26 were introduced into a pET-15b vector and recombinant proteins containing an N-(His)(6)-fusion tag were expressed in high yields. To resolve the problem of frameshift during expression of S26 caused by the presence of tandem arginine codons in its mRNA that are rare in Escherichia coli, we substituted the rare AGA codon with the more frequent arginine codon (CGC) using a primer with this mutation for PCR amplification of S26 cDNA. All proteins were expressed mainly in the form of inclusion bodies and purified to homogeneity by metal affinity chromatography in one step (except for S3). Expression of the full-length S3 was accompanied by the formation of a low molecular weight polypeptide that was co-purified with S3 by metal affinity chromatography. Complete purification of S3 required an additional gel-filtration step. The proteins were refolded by stepwise dialysis. Both identity and purity of the proteins were confirmed by 2D PAGE. The proteins obtained could be used in a wide range of applications in biophysics, biochemistry, and molecular biology.
Malygin
Alexey
A
Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Lavrentjev pr. 8, Novosibirsk 630090, Russia.
Baranovskaya
Oxana
O
Ivanov
Anton
A
Karpova
Galina
G
D001483:Base Sequence; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
C489203:RPS26 protein, human; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C029537:ribosomal protein S10; C040343:ribosomal protein S19; C044152:ribosomal protein S3; C030831:ribosomal protein S5