In Vitro Analysis Of Interaction Between Ribosome Protein L11 And Polypeptide Release Factor From Euplotes

Protein synthesis is one of the most important processes in organism, which includes the processes of initiation,elongation and termination.L11 is a highly conserved ribosomal protein essential for several steps in protein synthesis.The molecular switch,the N-terminal domain of L11 is responsible for the mechanism of EF-G-dependent translocation during the elongation cycle of protein synthesis.The N-terminal domain is also required for the functional interaction of release factor 1 with the ribosome in the translation termination.It is the target site for thiazole antibiotics including,antibiotics known to inhibit elongation factor-dependent ribosome activities,and has been shown to be important for stress activation of the transcription factor involved in the stringent response.To study the function of the L11 in eukaryotes,we cloned the gene encoding L11 protein from the macronuclear DNA of Euplotes octocarinatus. Recombinant plasmid pGEX-6p-1-L11 was constructed to express the GST fused protein Lll in E.coli.The fusion protein was purified using glutathion-Sepharose 4B affinity chromatography.The target protein was injected into mice to induce immunoreactions and the polyclonal antibody was prepared.Pull-down analysis showed that the recombinant GST-L11 interacted with the polypeptide chain release factor eRFla of E. octocarinatus.The result assumes that ribosomal protein L11 in lower eukaryotes may also play a role in translation termination.According to the reported amino acid sequence of the ribosome protein L11,two primers were designed and synthesized.The L11 gene fragment was amplified from the macronuclear DNA of Euplotes octocarinatus.The fragment was cloned and sequenced.Gene specific primers were then synthesized.Using Euplotes telomeric sequence(C₄A₄C₄A₄C₄A₄C₄) as primer in combination with the gene specific primer,additional L11 fragments were amplified and cloned.From these overlapping fragments the complete gene sequence coding for the L11 of E.octocarinatus was obtained. The macronuclear chromosome carrying the L11 gene is 709 bp long.It contains an open reading frame of 531 bp and encodes 176 amino acids. Characteristics of the gene and the deduced amino acid sequence were discussed.The accession number of the sequence in GenBank is EF061066.L11 was cloned into an expression vector pGEX-6p-1 and expressed as inclusion bodies in Escherichia coli BL21(DE3) host cells.The expression of E.coli BL21(DE3) / pGEX-6p-1-L11 was induced with IPTG.The inclusion body protein band in SDS-PAGE was excised and the protein,L11, was extracted.The protein was purified through GS4B Affinity Chromatography.Westem blot assay with anti-GST antibody confirmed that the protein is GST-L11.To remove GST tail,the fusion protein was digested by PreScission Protease.L11 was then injected into mice to induce immunoreactions,and the anti-sera were prepared.ELISA analysis showed that the titer for this polyclonal antibody was 1:8,000.Westem blot analysis showed that the antibody reacted specifically to expressed L11 protein,suggesting that the polyclonal antibody could be used for research of the function of the ribosome protein L11.In pull down assay,the fusion protein GST-L11 and His-eRFla were expressed in E.coli BL21(DE3) and purified by affinity chromatography, respectively.Supernatant containing recombinant His-eRFla was loaded into the GS4B beads that contain immobilized GST-L11.After incubation at 4℃, beads were washed to remove non-specific proteins.Bound proteins were eluted by adding of excess glutathione and analyzed by Western blot using anti-His antibody.Subsequently,it was confirmed that the GST-L11 could band to His-eRFla by using the same method.It used the Ni²⁺-NTA affinity chromatographic and the antibody of the Western blot was anti-GST.The results indicated the ribosome protein L11 and the Classâ… release factor eRFla from E.octocarinatus could interact in vitro.