Purification of human mitochondrial ribosomal L7/L12 stalk proteins and reconstitution of functional hybrid ribosomes in Escherichia coli

• Published in: Protein expression and purification Vol. 78; no. 1; pp. 48 - 54
• Main Authors: Han, Min-Joon, Cimen, Huseyin, Miller-Lee, Jennifer L., Koc, Hasan, Koc, Emine C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2011
• Subjects: Amino Acid Sequence; Animals; Blotting, Western; Cattle; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - genetics; Cell Cycle Proteins - isolation & purification; Cell Cycle Proteins - metabolism; Chromatography, Affinity; Duet expression; Elongation; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Homology; Humans; Hybrids; Mitochondria; Molecular Sequence Data; MRPL10; MRPL12; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - isolation & purification; Nuclear Proteins - metabolism; Potassium Chloride - chemistry; protein purification; Ribosomal Proteins - chemistry; Ribosomal Proteins - genetics; Ribosomal Proteins - isolation & purification; Ribosomal Proteins - metabolism; Ribosome; Ribosomes; Ribosomes - chemistry; Ribosomes - metabolism; Sequence Alignment; Solubility; Structure-function relationships; Translation; Translation elongation; Mitochondria; Translation; Ribosome; MRPL12; MRPL10; Duet expression
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2011.03.004

Bacterial ribosomal L7/L12 stalk is formed by L10, L11, and multiple copies of L7/L12, which plays an essential role in recruiting initiation and elongation factors during translation. The homologs of these proteins, MRPL10, MRPL11, and MRPL12, are present in human mitochondrial ribosomes. To evaluate the role of MRPL10, MRPL11, and MRPL12 in translation, we over-expressed and purified components of the human mitochondrial L7/L12 stalk proteins in Escherichia coli. Here, we designed a construct to co-express MRPL10 and MRPL12 using a duet expression system to form a functional MRPL10–MRPL12 complex. The goal is to demonstrate the homology between the mitochondrial and bacterial L7/L12 stalk proteins and to reconstitute a hybrid ribosome to be used in structural and functional studies of the mitochondrial stalk.