A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli.
With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3'-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)(6)-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)(6)-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.
Ederth
Josefine
J
Department of Cell and Molecular Biology, Uppsala University, S-751 24 Uppsala, Sweden.
Mandava
Chandra Sekhar
CS
Dasgupta
Santanu
S
Sanyal
Suparna
S
D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods*; D004151:Dipeptides / Q000096:biosynthesis; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics*; D005818:Genetic Engineering; D006639:Histidine / Q000737:chemistry / Q000235:genetics*; D007093:Imidazoles / Q000737:chemistry; D008164:Luminescent Proteins / Q000096:biosynthesis; D009842:Oligopeptides / Q000235:genetics*; D014176:Protein Biosynthesis; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics*; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism
D004151:Dipeptides; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D007093:Imidazoles; D008164:Luminescent Proteins; D009842:Oligopeptides; D012269:Ribosomal Proteins; C512844:rplL protein, E coli; D006639:Histidine; C029899:imidazole