Leontiadou F, Tsagkalia A, Choli-Papadopoulou T
Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.
Amino Acids. 2007 Sep;33(3):463-8. doi: 10.1007/s00726-006-0438-3. Epub 2006 Oct 18.
Protein L4 from the thermophilic bacterium Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells and purified under native conditions by using ion exchange chromatography. Although it's known strong binding to RNA (23S rRNA as well as mRNA) the yield of the purified protein was 6 mg per 10 g of cells and it is similar to that referred for Thermotoga maritima L4 ribosomal protein. In addition, E. coli cells harboring the wild type Thermus thermophilus L4 (wtTthL4) ribosomal protein as well as its mutant having changed the highly conserved glutamic acid 56 by alanine (TthL4-Ala 56) were incorporated into E. coli ribosomes after transformation of the host cells with the recombined vector. The cells having incorporated the mutant TthL4-Ala56 are more sensitive against erythromycin related to that containing the wtTthL4 protein.The resistance to the drug indicates that the mutated amino acid Glu56 is probably critical for the local ribosomal conformation and that its mutation induces conformational disturbances that are "transferred" to the entrance of the major exit tunnel, the place where the drug does bind.
嗜热栖热菌（Thermus thermophilus）的L4蛋白（TthL4）在大肠杆菌细胞中进行了异源过量表达，并通过离子交换色谱法在天然条件下进行了纯化。尽管已知它能与RNA（23S rRNA以及mRNA）强烈结合，但纯化后的蛋白产量为每10克细胞6毫克，这与嗜热栖热放线菌（Thermotoga maritima）L4核糖体蛋白的产量相似。此外，在用重组载体转化宿主细胞后，携带野生型嗜热栖热菌L4（wtTthL4）核糖体蛋白及其将高度保守的谷氨酸56突变为丙氨酸的突变体（TthL4-Ala 56）的大肠杆菌细胞被整合到大肠杆菌核糖体中。与含有wtTthL4蛋白的细胞相比，整合了突变体TthL4-Ala56的细胞对红霉素更敏感。对该药物的抗性表明，突变的氨基酸Glu56可能对局部核糖体构象至关重要，其突变会诱导构象紊乱，这种紊乱会“传递”到主要出口通道的入口处，即药物结合的位置。
