Study On Effect Of The Nuclear Localization Signal Domain On Importing P33ING1b Into Nucleus As Well As Expression And Purification Of Its GST-NLS Fusion Protein

Objective Inhibitor of growth gene 1 (ING1 gene) found in 1996 locates in13q33-34 of human chromosome. As a growth inhibitor gene, its principal physiological function is regulating cell cycle, inhibiting cell proliferation, as well as plays also the important roles on regulating cell senescence and apoptosis, and maintaining stabilization of genome. The p33^ING1b is its main expression protein. The past studies showed that mutation rate of the gene was very low in the most malignant tumors, but that levels of its mRNA transcription and protein expression have no uniform conclusion. However, a significant finding is p33^ING1b subcellular mislocalization in human glioma cells. In other word, p33^ING1b locates in the tumor cytoplasm instead of in nucleus as normal cells. This abnormity is possibly an important factor which results in pathogenesis and progression of human gliomas. The proteins from cytoplasm into nucleus are usually transported by importin α/β system. Not only does importin a, as an adaptor, bind to nuclear localization signal domain (NLS) of transported proteins, but also does to importin p. Through the interaction between importin β and nuclear pore complex, the process transporting proteins into nucleus is started. However, how does p33^ING1b enter nucleus in normal cells? What is the cause which results in p^ING1b subcellular mislocalization in glioma cells? Both their mechanisms are not still known so far and should be further researched.Therefore, starting with function study of p33^ING1b NLS, we preliminarily investigated the physiological effect of p33^ING1b NLS on transporting p33^ING1b from cytoplasm into nucleus by a series of molecular biological methods, in order to provide the important clues and the useful research tools for exploring the mechanisms of p33^ING1b into nucleus in normal cells and p33^ING1b subcellular mislocalization in glioma cells.Methods In the first part of this study, firstly c-DNA of pSS^0111 NLS wasgained from human embryo lung fibroblast line (MRC-5 cells) by RT-PCR. Then the c-DNA was inserted into clone vector (Pbluescript-SK) by gene recombination technology. After its sequence correctness had been identified, the c-DNA was inserted into multiple clone site of expression vector (pEGFP-Cl), in order to construct recombined eukaryotic expression vector (pEGFP-Cl-NLS) of fusion protein (GFP-NLS) of green fluorescence protein(GFP) and pSS?0113 NLS. pEGFP-Cl-NLS was then transfected into MRC-5 cells to express GFP-NLS. Via tracer effect of GFP, subcellular localization of GFP-NLS was observed under fluorescence microscope, in order to prove whether pSS?0113 NLS played an important role on transporting pSS15"1011* into nucleus in physiological condition.In the second part of this study, using c-DNA of pSS?016 NLS in Pbluescript-SK as template, the NLS c-DNA sequence to insert fittingly into multiple clone site of pGEX-5X-3 was obtained by PCR amplification. Then the c-DNA sequence was inserted into multiple clone site of pGEX-5X-3 to construct recombined prokaryotic expression vector (pGEX-5X-3-NLS) of fusion protein (GST-NLS) of glutathion-S-transferase (GST) and pSS^011* NLS. Finally, this vector was transducted into E.coli BL21 and it expressed highly GST-NLS under IPTG inducing. The E.coli BL21 which expressed GST-NLS was split by ultrasonic and the splitting lysate was mixed with glutathione-sepharose beads. GST-NLS was pulled down by the specific binding between GST and glutathione. After impure proteins had been scoured off, GST-NLS was eluted down with eluting buffer.ResultsThe first part: The assays of enzyme cutting and DNA sequencing showed that c-DNA sequence of p33INGlb NLS had been successfully obtained from MRC-5 cells by RT-PCR and inserted into multiple clone site of Pbluescript-SK. The sequencing analysis confirmed that the c-DNA was amplified correctly in Pbluescript-SK. After the amplified c-DNA had been inserted into eukaryotic expression vector pEGFP-Cl, the base sequences of pSS"1"3115 NLS, its reading frame and stop codon were detected by enzyme cutting and DNA sequencing. All of them were completely exactitude.After the recombined vector pEGFP-Cl-NLS was transfected into MRC-5 cells, GFP-NLS was effectively expressed and its green fluorescence signal completely located in the nucleus. However, the GFP fluorescence signal expressed by vacant vector pEGFP-Cl in MRC-5 cells located completely in the cytoplasm.The second part: The identify of enzyme cutting and DNA sequencing revealed that we had successfully obtained c-DNA sequence of pSS^0113 NLS from Pbluescript-SK by PCR amplification and successfully inserted it into multiple clone site of pGEX-5X-3. The analysis of enzyme cutting and DNA sequencing validated that base sequence of the inserted c-DNA was completely correct and its length was 183 bp. The analysis of reading frame indicated that GST expressed by vacant vector pGEX-5X-3 consisted of 243 aminophenols and its molecular weight was 26.73kDa, and GST-NLS expressed by recombined vector pGEX-5X-3-NLS contained 287 aminophenols, the molecular weight 31.57kDa. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that an amount of the GST-NLS and GST had been successfully expressed in E.coli BL21 by IPTG inducement. Both GST-NLS and GST were successfully purified by the affinity deposition of glutathione-sepharose beads. SDS-PAGE did not display the obvious bands of impure proteins in both purified GST-NLS and GST.Conclusion Above results prove that in physiological state, p33INGlb locatesabsolutely in nuclei of living cells and its NLS plays a decisive role when it is transported into nucleus. Moreover, in this study, we successfully constructed the recombined expression vectors of GFP-NLS and GST-NLS, established relevant cell experiment models, and a series of experiment technologies expressing highly GST-NLS fusion protein and GST protein and purifying them. All of them provide the very practical research tools for further exploring the physiological mechanism of jnt0 nucjeus ancj ajso i^ a solid foundation for searching the causes of gybcejjujaj. mislocalization in glioma and the other malignant tumor cells.